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A unifying paradigm for transcriptional heterogeneity and squamous features in pancreatic ductal adenocarcinoma

Nature Cancer volume 1, pages 59–74 (2020)Cite this article

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Abstract

Pancreatic cancer expression profiles largely reflect a classical or basal-like phenotype. The extent to which these profiles vary within a patient is unknown. We integrated evolutionary analysis and expression profiling in multiregion-sampled metastatic pancreatic cancers, finding that squamous features are the histologic correlate of an RNA-seq-defined basal-like subtype. In patients with coexisting basal and squamous and classical and glandular morphology, phylogenetic studies revealed that squamous morphology represented a subclonal population in an otherwise classical and glandular tumor. Cancers with squamous features were significantly more likely to have clonal mutations in chromatin modifiers, intercellular heterogeneity for MYC amplification and entosis. These data provide a unifying paradigm for integrating basal-type expression profiles, squamous histology and somatic mutations in chromatin modifier genes in the context of clonal evolution of pancreatic cancer.

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Fig. 1: Study overview and morphologic heterogeneity for squamous features in PDAC.
Fig. 2: Transcriptional heterogeneity for SF in end-stage PDAC.
Fig. 3: Expressional profiles based on three major classification schemes in end-stage PDAC.
Fig. 4: Genomic landscape of end-stage PDAC with and without SF.
Fig. 5: Integration of transcriptomic and morphologic features with phylogenetic patterns in PDAC PAM55 with clonal KMT2C mutation.
Fig. 6: Integration of transcriptomic and morphologic features with phylogenetic patterns in PDAC PAM02 with clonal ARID1A mutation.
Fig. 7: Integration of transcriptomic and morphologic features with phylogenetic patterns in PDAC PAM46 with MYC amplification.
Fig. 8: Squamous features in pancreatic ductal adenocarcinoma correspond to enhancement of MYC.

Data availability

RNA and DNA sequence data for this study have been deposited at the European Genome-phenome Archive under accession number EGAS00001003974. Published gene sets analyzed here are available from previous papers6,10,11. Sequencing data from the MSK IMPACT cohort that were analyzed here18 are publicly available at cBioPortal (https://www.cbioportal.org/). The other human resected pancreatic cancer data were derived from TCGA Research Network: http://cancergenome.nih.gov/. The dataset derived from this resource that supports the findings of this study is available through Firebrowse (http://firebrowse.org/). Source data for Figs. 1, 3, 4, 8 and Extended Data Figs. 2–4 and 10 have been provided as Source Data Figs. 1, 3, 4 and 8 and Source Data Extended Data Figs. 2–4 and 10. All other data supporting the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

We are grateful to G. Askan, A. Yavas and J.V. Egger for assistance in identifying resected adenosquamous samples for use in this study, to S. Yamamoto for analysis tool information and to S. Oki for technical support. We gratefully acknowledge the members of the Molecular Diagnostics Service in the Department of Pathology for MSK IMPACT. This work was supported by National Institutes of Health grant nos. R01 CA179991 and R35 CA220508 to C.I.D., F31 CA180682 and 2T32 CA160001-06 to A.M.M. and CA62924 to R.H.H., the Daiichi-Sankyo Foundation of Life Science Fellowship to A.H., the Mochida Memorial Foundation for Medical and Pharmaceutical Research Fellowship to A.H., Cycle for Survival and the Marie-Josée and Henry R. Kravis Center for Molecular Oncology. MSK IMPACT was funded in part by the Marie-Josée and Henry R. Kravis Center for Molecular Oncology and the National Cancer Institute Cancer Center Core grant no. P30-CA008748.

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Authors and Affiliations

  1. The David M. Rubenstein Center for Pancreatic Cancer Research, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Akimasa Hayashi, Jun Fan, Alvin P. Makohon-Moore, Nicolas Lecomte, Yi Zhong, Jungeui Hong, Jinlong Huang, Hitomi Sakamoto, Marc A. Attiyeh, Zachary A. Kohutek, Lance Zhang, Aida Boumiza, Rajya Kappagantula, Priscilla Baez, Jessica Bai, Jerry P. Melchor, Eileen M. O’Reilly, David S. Klimstra & Christine A. Iacobuzio-Donahue

  2. Human Oncology and Pathogenesis Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Akimasa Hayashi, Jun Fan, Alvin P. Makohon-Moore, Nicolas Lecomte, Yi Zhong, Jungeui Hong, Jinlong Huang, Hitomi Sakamoto, Marc A. Attiyeh, Zachary A. Kohutek, Lance Zhang, Aida Boumiza, Rajya Kappagantula, Priscilla Baez, Jessica Bai, Jerry P. Melchor & Christine A. Iacobuzio-Donahue

  3. Cell Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Ruoyao Chen & Michael Overholtzer

  4. Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Yu-jui Ho, Marta Lisi, Kalyani Chadalavada & Gouri J. Nanjangud

  5. Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Winston Wong & Eileen M. O’Reilly

  6. Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Olca Basturk, David S. Klimstra & Christine A. Iacobuzio-Donahue

  7. Department of Pathology, Sol Goldman Pancreatic Cancer Research Center, The Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Ralph H. Hruban & Laura D. Wood

Authors
  1. Akimasa Hayashi
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  2. Jun Fan
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  3. Ruoyao Chen
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  4. Yu-jui Ho
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Contributions

A.H. and C.A.I.-D. designed the study. A.H., J.F., A.P.M.-M., H.S., M.A.A., A.B., R.K., P.B., L.D.W., R.H.H. and C.A.I.-D. collected autopsy samples. A.H. and C.A.I.-D. reviewed the histology of autopsy samples and selected cases. O.B., D.S.K., A.H. and C.A.I.-D. reviewed the pathology of MSK Clinical IMPACT cases. A.H. and C.A.I.-D. reviewed the pathology of surgical cases in TCGA cohort. A.H., R.C., M.O., K.C., M.L., G.J.N. and C.A.I.-D. reviewed the entosis of Immuno-FISH slides. A.H. and J.F. prepared RNA samples. A.P.M.-M., J.Hong, H.S., Z.A.K. and A.H. prepared the DNA samples. A.H., Y.Z. and C.A.I.-D. performed RNA sequencing. Y.H., A.H., L.Z. and J.Huang analyzed RNA sequencing results. A.P.M.-M., J. Ho., Z.A.K., H.S. M.A.A., A.H., and C.A.I.-D. performed DNA sequencing. M.A.A., A.P.M.-M., J.Hong, A.H. and C.A.I.-D. analyzed DNA sequencing results and derived the phylogenies. A.P.M.-M., J.Hong, A.H., J.P.M. and C.A.I.-D. managed the sequencing data. W.W. and E.M.O. collected samples and clinical information for MSK Clinical IMPACT. M.L., K.C. and G.J.N. performed immuno-FISH. J.B. and N.L. performed organoid experiments. A.H., R.C., Y.H., M.O., N.L. and C.A.I.-D. wrote the manuscript. All authors reviewed and edited the final manuscript.

Corresponding author

Correspondence to Christine A. Iacobuzio-Donahue.

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Competing interests

D.S.K. is a consultant and equity holder to Paige.AI and a consultant to Merck Pharmaceuticals and receives royalties from UpToDate and the American Registry of Pathology.

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Extended Data

Extended Data Fig. 1 Case Selection and Postmortem Diagnosis.

(a) Schematic of case selection for current study. (b) Immunolabelling for glandular and squamous or squamoid components in 13 Representative PDACs. All regions with squamous differentiation (SD) showed positivity for CK5/6 and p63, whereas no labeling was observed in regions with glandular morphology (GL). In two PDACs with the neoplastic cells stained positive for CK5/6 but were negative for p63 and thus classified as having squamoid features. n.d., no immunolabeling performed. IHC was done for 31 case, 167 slides. Scale bar: 100 um. (c) Postmortem case diagnoses. Seven cases corresponded to adenosquamous carcinoma (ASC), two cases showed squamoid features (SF) and four cases showed focal (<30%) squamous differentiation (SD).

Extended Data Fig. 2 Squamous Features of Pancreatic Ductal Adenocarcinoma in the TCGA and MSK Clinical IMPACT Patient Cohorts.

(a) Schematic for histological classification of cases in the TCGA and MSK clinical IMPACT cohorts. (b) Schematic for case selection in the TCGA and MSK clinical IMPACT cohorts. (c) Frequency of case diagnoses in TCGA (n = 145), MSK Clinical IMPACT (n = 617) and our autopsy (n = 123) cohort. (d) Representative digital images of adenosquamous carcinoma (ASC) (out of 3 cases), PDAC with potential squamoid feature or squamous differentiation (PDAC with potential SF/SD) (histologic diagnosis modified based on our re-review) (out of 9 cases), and poorly differentiated ductal adenocarcinoma (out of 57 cases) in TCGA. Alveolar or trabecular pattern was confirmed in ASC or PDAC with potential SF/SD. (e) Kaplan-Meier analysis showed poor prognosis of ASC or PDAC with potential SF/SD (n = 12) compared to conventional PDAC (n = 129) in TCGA cohort (P < 0.0001, Log-rank test). (f) Kaplan-Meier analysis showed poor prognosis of ASC or PDAC with potential SF/SD (n = 70) compared to conventional PDAC (n = 494) in MSK-IMPACT cohort (P = 0.001, Log-rank test).

Source data

Extended Data Fig. 3 Squamous Feature Associated Alteration and Characteristic in Pancreatic Ductal Adenocarcinoma.

(a) Entotic CICs in matched glandular (GL) versus squamoid or squamous morphology (SF/SD) in 10 patients. Statistics are performed using Mann–Whitney U test, two-sided. (62, 62, 73, 38, 50, 26, 33, 21, 8 and 21 blocks/slides were used for entosis evaluation of PAM02, MPAM06, PAM73, PAM55, PAM22, PAM28, PAM53, PAM80, PAM20 and PAM39). (b) mRNA Expression of squamous markers (TP63, KRT5 and KRT6A) in samples with glandular growth pattern (GL) (n = 133), squamoid features (SF) (n = 18) and squamous differentiation (SD) (n = 63). SF have intermediate expression pattern between SD and GL. Each P-value is calculated by Mann–Whitney U test, two-sided. Lines and bars: median with interquartile range. (c) mRNA expression of TP63, KRT5 and KRT6A. ASC (n = 3) and PDAC with potential SF/SD (n = 9) have higher expression of TP63 than conventional PDAC (n = 133) in TCGA (P = 0.007, Mann–Whitney U test, two-sided). (d) Keratin network based on mRNA expression. In GL, KRT19 (normally expressed in ductal epithelia) is a hub in pancreas cancer. In SF, KRT6A and KTR5 (normally expressed in squamous epithelium) have some interaction. In SD, stratified squamous epithelium keratins (KRT4, KRT5, KRT13, KRT14) and heavy weight keratins (KRT1 and KRT10) are expressed in the network. (e)-(g) Tumor purity in PDACs with or without squamous feature. (e) Tumor purity by FACETs in end stage PDAC. Samples with squamous differentiation (SD) (n = 43) have higher tumor purity than samples with squamoid feature (SF) (n = 20) or glandular pattern (GL) (n = 152) (P = 0.012 or P < 0.001, Mann–Whitney U test, two-sided). Lines and bars: median with interquartile range. (f) Intratumoral heterogeneity of tumor purity in end stage PDAC. Samples with SF or SD have higher tumor purity in one tumor (9, 11, 8, 5, 6, 11, 5, 9, 4, 12, 6, 9, 21, 8, 14, 8, 10, 7, 11, 8, 3, 7, 9, 6 and 8 samples were used for PAM46, MPAM06, PAM54, PAM53, PAM32, PAM02, PAM28, PAM22, PAM16, PAM55, PAM20, PAM39, PAM52, PAM48, PAM24, PAM56, PAM51, PAM49, PAM03, PAM29, PAM25, PAM47, PAM50, PAM27, and PAM04) (g) Absolute tumor purity in TCGA cohort. Absolute tumor purity is not different between conventional PDAC (n = 132) and PDAC with potential SF/SD (n = 9) and ASC (n = 3) (P = 0.601, Mann–Whitney U test, two-sided).

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Extended Data Fig. 4 Mutational Characteristics of the MSK clinical IMPACT cohort.

(a) Oncoprint illustrating somatic alterations of chromatin modifier genes, RB1 and MYC amplification in 617 PDAC cases including 26 ASCs and 51 PDACs with potential SF/SD. P-value was tested using two-sided Fisher’s exact test. * indicates P-value if analysis is confined to driver gene mutations only. (b) Entotic CIC are more frequent occur in TP53 mutant PDACs (n = 180) than in TP53 wild type PDACs (n = 42) (P = 0.021, Mann–Whitney U test, two-sided).

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Extended Data Fig. 5 Integration of Transcriptomic and Morphologic Features with Phylogenetic Patterns in Pancreatic Ductal Adenocarcinoma (a-d) PAM54 with clonal KMT2C mutation and (e-h) PAM16 with clonal KDM6A mutation.

(a) Phylogenetic analysis illustrating the clonal relationship of samples analyzed in this patient. The predicted timing of somatic alterations in driver genes and whole genome duplication are also shown. Mutations in chromatin modifier genes are in red font, all others in orange. Clonal driver genes are notable for a KMT2C somatic alteration, whereas mutations in RB1 and SMARCA4 (two independent mutations) are present in a subset of samples. SD in this carcinoma was found in all samples analyzed, although it was admixed with a minor glandular component in some samples. (b) Principal components analysis (214 samples from 27 patients) highly similar expression between samples (all SD, n = 18) in PAM54. (c) Relationship of anatomic location to morphologic and transcriptional profiles. (d) Representative histologic images of tumors (out of total 108 histologic images for PAM54) in the same patient. Scale bar: 100um. (e) Clonal driver genes are notable for a KDM6A somatic alteration. SD in this carcinoma was found in all samples analyzed, although it was admixed with a minor GL component in some samples. (f) Principal components analysis (214 samples from 27 patients) illustrates highly similar expression between samples (all SD, n = 4) in PAM16. (g) Relationship of anatomic location to morphologic and transcriptional profiles. (h) Representative histologic images of metastatic tumors PT3 and PT4 (out of total 15 histologic images for PAM16). Scale bar: 100um.

Extended Data Fig. 6 Integration of Transcriptomic and Morphologic Features with Phylogenetic Patterns in Pancreatic Ductal Adenocarcinoma (a)-(d) PAM39 and (e)-(g) PAM20 with clonal ARID1A mutation.

(a) Phylogenetic analysis illustrating the clonal relationship of samples analyzed in this patient. The predicted timing of somatic alterations in driver genes, whole genome duplication and MYC amplification are shown. Mutations in chromatin modifier genes are in red font, all others in orange. Red outline indicates the one sample with SF based on histology and immunohistochemical analysis (squares) but a classical type expression profile (triangle). Clonal driver genes are notable for an ARID1A somatic alteration. SF is confined to one prostate metastasis sample (PT9). (b) Principal components analysis (214 samples from 27 patients) shows a similar gene expression profile between the samples with GL (n = 7) or SF (n = 1) morphology in PAM39. (c) Relationship of anatomic location to morphologic and transcriptional heterogeneity. (d) Representative histologic and/or immunohistochemical images of the primary (PT1) and metastasis (PT6, PT8, PT9) tumors (out of total 21 histologic images for PAM39). Scale bar: 100um. (e) Clonal driver genes are notable for an ARID1A somatic alteration. MYC amplification (≥ 6 copies) was detected in all samples with SF, and in a phylogenetically distinct sample with GL within the primary tumor. Samples with SF in this carcinoma (PT3-PT6) are clonally related. (f) Relationship of anatomic location to morphologic heterogeneity. The metastasis samples PT3-PT6 showed SF whereas the primary tumor samples showed GL. (g) Representative histologic and/or immunohistochemical images of the primary tumor (PT1) and diaphragm metastasis (PT5) (out of total 8 histologic images for PAM20). Scale bar: 100um.

Extended Data Fig. 7 Integration of Transcriptomic and Morphologic Features with Phylogenetic Patterns in Pancreatic Ductal Adenocarcinoma (a)-(d) PAM28 and (e)-(h) MPAM6 with clonal RB1 Mutation.

(a) Phylogenetic analysis illustrating the clonal relationship of samples analyzed in this patient. The predicted timing of somatic alterations in driver genes and whole genome duplication are shown. The mutation in RB1 is in red font, all others in orange. Purple outline indicates samples that have SD based on histology (squares). Clonal driver genes are notable for an RB1 somatic alteration. Samples with SD are more related to each other than to other samples in this patient. (b) Principal components analysis (214 samples from 27 patients) shows that samples PT1-PT3 with basal-like type expression and SD morphology (n = 5) are distinct from samples PT4 and PT5 that have basal-like type expression but GL morphology (n = 2) in PAM28. (c) Relationship of anatomic location to morphologic and transcriptional heterogeneity. Both GL and SF/SD were seen in the primary tumor, yet liver metastases PT4 and PT5 have GL morphology and a basal-like type expression profile. (d) Representative histologic images and immunohistochemical labeling of primary tumor sample PT1 and liver metastases PT4 and PT5 (out of total 26 histologic images for PAM28). Scale bar: 100um. (e) Clonal driver genes are notable for a deleterious RB1 mutation. Samples with SD (PT5-PT7) are more related to each other than to other samples in the same patient. (f) Principal components analysis (214 samples from 27 patients) indicates distinct gene expression profiles between GL (n = 8) and SD (n = 2) samples in MPAM06. (g) Relationship of anatomic location to morphologic and transcriptional heterogeneity. SD is confined to the liver metastases (PT5-PT7). (h) Representative histologic images of the primary and multiple metastatic tumors in the same patient (out of total 62 histologic images for MPAM6). Scale bar: 100um.

Extended Data Fig. 8 Integration of Transcriptomic and Morphologic Features with Phylogenetic Patterns in Pancreatic Ductal Adenocarcinoma (a)-(d) PAM22 and (e)-(h) PAM53.

(a) Phylogenetic analysis illustrating the clonal relationship of samples analyzed in this patient. Purple outline indicates samples that have SD based on RNAseq (triangles) and histology/immunohistochemistry (squares). The predicted timing of somatic alterations in driver genes, whole genome duplication and MYC amplification are also shown. SD is confined to a single sample within the multiregion sampled primary tumor (PT2). (b) Principal components analysis (214 samples from 27 patients) indicates that SD (n = 2) including PT2 show a different expression profile from all other primary tumor samples that have GL (n = 7) morphology in PAM22. (c) Relationship of anatomic location within the primary tumor to morphologic and/or transcriptional heterogeneity for SF/SD. (d) Representative histologic images of representative tumors in the same patient (out of total 50 histologic images for PAM22). Scale bar: 100um. (e) The one sample with a classical expression profile and GL (PT3) morphology forms the outgroup in the tree. Four samples with basal-like expression and SD correspond to both the primary tumor (PT4 and PT5) and metastasis (PT1 and PT2). (f) Principal components analysis (214 samples from 27 patients) indicates samples PT1 and PT3 have relatively different expression profiles from other SD samples (total 18 samples) in PAM53. (g) Relationship of anatomic location to morphologic and transcriptional heterogeneity. SD was found in one omental metastasis (PT3) which is also showed basal-like expression. (h) Representative histologic and immunohistochemical images of the primary tumor samples PT4 and PT5, liver metastasis PT1 and omental metastasis PT3 (out of total 33 histologic images for PAM22). Scale bar: 100um.

Extended Data Fig. 9 Morphologic Features with Phylogenetic Patterns in Pancreatic Ductal Adenocarcinoma PAM32.

(a) Phylogenetic analysis illustrating the clonal relationship of samples analyzed in this patient. The predicted timing of somatic alterations in driver genes and whole genome duplication are also shown. Purple outline indicates samples that have SD based on histology and immunolabeling (squares). Samples PT3-PT6 with SD are more closely related to each other than to other samples in the same patient. (b) Relationship of anatomic location to morphologic heterogeneity. One liver metastasis (PT7, not sequenced) showed GL morphology. (c) Representative histologic images of the primary and metastatic tumors in this patient (out of total 39 histologic images for PAM32). Scale bar: 100um.

Extended Data Fig. 10 Molecular Characteristics in Squamous Feature and Entosis.

(a)-(d) Impact of MYC-overexpression Using PDAC Organoid Models. (a) Overexpression of MYC and alteration status of chromatin modifier genes in eight PDAC organoids. Center value and bar: mean and SD. Three data points in each organoid means technical triplicates of qPCR data. (b) Representative images of PDAC organoids (out of 8 organoids, 64 images). Images of organoids were acquired 5 days post-sorting (10 days total post-infection). No obvious morphological changes were identified between the MYC-infected vs the mock-infected organoids. Scale bar: 50um. (c) Relative mRNA expression of squamous markers (TP63, KRT5 and KRT6A) after MYC overexpression. Two PDAC organoids with chromatin modifier mutations (HT160c and HT28) shows higher expression of all three markers whereas no effects are seen in the absence of mutations in these genes. Center value and bar: mean and SD. Three data points in each organoid means technical triplicates of qPCR data. **Appropriate KRT5 in HT151 signal was not detected with qPCR due to low expression. (d) Metabolic pathways are enriched in Entotic cases based on five different databases. (e) MYC expression in Entotic CIC. Winner (MYC positive)- Loser (MYC negative) pattern was identified both MYC amplified and non-amplified cases. W(P)-L(P), W(P)-L(N), W(N)-L(P) and W(N)-L(N) are Winner (MYC positive)-Loser (MYC positive), Winner (MYC positive)-Loser (MYC negative), Winner (MYC negative)-Loser (MYC positive) and Winner (MYC negative)-Loser (MYC negative) patterns respectively. (f) Representative image of Winner (positive)- Loser (negative) pattern (out of 9, 6, 1 and 14 images in W(P)-L(P), W(P)-L(N), W(N)-L(P) and W(N)-L(N) patterns).

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Supplementary information

Supplementary Tables 1–10

Reporting Summary

Supplementary Dataset 1

S1 RNA expression of each sample (log2 conversion of DESeq2 normalized data).

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Hayashi, A., Fan, J., Chen, R. et al. A unifying paradigm for transcriptional heterogeneity and squamous features in pancreatic ductal adenocarcinoma. Nat Cancer 1, 59–74 (2020). https://doi.org/10.1038/s43018-019-0010-1

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