Tumor-Infiltrating Leukocyte Phenotypes Distinguish Outcomes in Related Patients With Pancreatic Adenocarcinoma.

INTRODUCTION

Pancreatic ductal adenocarcinoma (PDAC) carries one of the highest mortality risks of all cancers. Most patients present with either metastatic PDAC (50%-60%) or locally advanced tumors (30%-40%), for which the median survival is 5-9 months after diagnosis.[1,2] Outcomes are still suboptimal for the small subset (10%-20%) of patients who present with resectable tumors confined to the pancreas; < 50% of these patients survive 5 years after surgery despite modern adjuvant chemotherapy.[3] Moreover, at autopsy, the disease is metastatic for 88% of recurrences, and > 80% have more than 10 distinct metastatic lesions[4] that are genetically related to the primary tumor[5] and other metastases.[6] These observations indicate that the majority of patients with PDAC harbor nonradiographically evident, micrometastatic disease after resection and that remnant tumor cells evade—or acquire resistance to—adjuvant chemotherapy. Although tropism to specific organs during metastatic spread is still poorly understood, patients with recurrence in the liver or peritoneum[7] survive significantly shorter than patients with recurrent lung metastases.[8-10]

PDAC tumors are commonly classified into two major subtypes: one that shares some features with adenosquamous tumors (squamoid and/or basal) and the other that retains a differentiated, glandular and/or ductal morphology (ductal and/or classical). Squamoid PDAC tumors are more glycolytic,[11,12] more hypoxic,[13] more likely to metastasize,[14,15] recruit inflammatory fibroblasts,[16] and yield a poorer prognosis than ductal tumors.[17-21] Classical subtype tumors are associated with more tumor-infiltrating leukocytes, denser collagen, and better outcomes,[15,22] although significant subtype heterogeneity within tumors[13] complicates the relationship between subtype and outcome.

More cytolytic T cells infiltrate PDAC tumors than most other tumors,[23] but they are ultimately insufficient to control the cancer. Density of tumor-infiltrating T cells is highly variable among PDAC tumors,[24] and abundant CD8+ T cells (along with a high number of neoantigens) can support exceptionally long-term survival.[25] However, T cell–mediated tumor immunity may be restricted by several mechanisms: tumor cell downregulation of HLA, immunosuppressive leukocytes, T cell proximity to tumor cells, and T cell exhaustion.[26-30] Effective T cell–mediated tumor immunity is enhanced by a high diversity of functional T cell clones, tumor-specific recruitment of effector T cells, and abundant neoantigens.[23,25,31] Quantifying the phenotypes, functions, and locations of leukocytes is critical to identify specific tumor immunity required for exceptional positive outcomes.

Rare, exceptional control of PDAC progression may be mediated by equally rare, idiosyncratic patient genetics, tumor-specific somatic alterations and gene expression, and/or stochastically generated tumor immunity. In this report, we present the cases of Pt1, who survived more than 46 months with occult, chemotherapy-resistant metastatic PDAC, and her niece (Pt2) who succumbed to progressing metastatic disease despite aggressive treatment. We compare the clinical outcomes and primary and metastatic tumors from these two patients, identifying major differences in subtype, somatic alterations, and leukocyte lineages that—in some combination or alone—might have led to divergent disease courses.

RESULTS

Disparate Outcomes for Two Related Patients Both Diagnosed With Stage II PDAC

Pt1 is a chronic smoker diagnosed at 64 years of age with stage II PDAC (pT3 N1 M0) and treated 26 days later by surgical resection of a moderately to poorly differentiated, invasive ductal adenocarcinoma, followed by two cycles of gemcitabine and capecitabine. A pulmonary lesion measuring 20 mm × 16 mm was evident by CT scan 27 days prior to resection of the primary tumor, but upon biopsy was negative for malignancy. However, during adjuvant chemotherapy, the lung lesion increased to 23 mm × 18 mm, and a CT and/or PET was suspicious for malignancy with a standardized uptake value (SUV) of 4.5. Based on the imaging, the lung lesion was resected and found to be metastatic PDAC. Pt1 chose to receive no further treatment and was radiographically disease-free for over 2 years (Fig 1) until an isolated, recurrent lung metastasis was identified by CT and FDG-PET (SUV of 9) and then treated by 50 Gy in five fractions using Stereotactic Body Radiotherapy. Four months later, restaging CT revealed that the size of the previously visualized left lower lobe nodule had decreased; there were no additional suspicious lesions, and the patient is asymptomatic 46 months after diagnosis.

FIG 1.

Disease courses for patients 1 (top) and 2 (bottom) aligned by date of diagnosis. The date when metastatic disease was first radiographically evident is indicated with an M in blue. CA19-9 test results are not shown because—for both patients—they were reproducibly approximately 90% lower than the 37 U/mL threshold for normal, even when the primary tumor was present. This result suggests a false negative result because of a Lewisa-b- antigen phenotype, as is the case for 5%-10% of Caucasian patients with PDAC.[65]

Pt2—a nonsmoker—was diagnosed at 44 years of age with stage II PDAC (pT3 N1 M0) and treated by surgical resection of a moderately differentiated adenocarcinoma with squamoid features as described by Hayashi et al[32]—this tumor was not qualified as an adenosquamous subtype of ductal adenocarcinoma. Pt2 was treated with six cycles of gemcitabine and nab-paclitaxel (on a clinical trial). A biopsy-proven, right ureter metastasis in the context of widespread peritoneal metastases was identified 1 year after the last dose of adjuvant chemotherapy, and Pt2 was treated with four cycles of fluorouracil and oxaliplatin. Subsequently, a right retroperitoneal metastasis and multiple liver metastases were identified and continued to progress despite further treatment (Fig 1). Pt2 died 19 months after metastatic disease recurrence.

A Shared Germline PRSS1 Mutation and Somatic Alterations Common to PDAC

We identified germline PRSS1 p.A16V in both patients and somatic alterations in the two most common PDAC driver genes—KRAS and TP53—in both tumors from each patient (Table 1). The primary tumor from Pt2 also contained a CDKN2A alteration. Metastatic tumors from both patients had all alterations found in their respective primary tumors. Additionally, the Pt1 metastasis contained an FANCA mutation, whereas the Pt2 metastasis contained a PTEN deletion. SMAD4 was altered in both the primary and metastatic tumors from Pt1, but only in the metastasis from Pt2.

TABLE 1.

Known PDAC Driver Genes Altered in the Primary and Metastatic Tumors From Pt1 and Pt2

Evidence of Squamoid Differentiation in Tumors From Patient 2

The histology of Pt1's primary tumor was homogeneously ductal and did not express KRT5, whereas Pt2's primary tumor contained heterogeneous squamoid features and KRT5 expression in approximately 25% of tumor cells (Fig 2A).[32] The lung metastasis from Pt1 was ductal, whereas the ureter metastasis from Pt2 was mostly squamoid (Fig 2B).

FIG 2.

PDAC subtype assignment based on histology and gene expression. (A) H&E stained sections and immunohistofluorescence for KRT5 (blue) and DAPI (white) from primary tumors. Regions of ductal or squamoid histology are indicated by black or blue arrows, respectively. Each scale bar is 0.5 mm. (B) H&E stained sections of metastases from Pt1 (lung) and Pt2 (ureter). Each scale bar is 0.5 mm. (C) PDAC subtyping of each patient's primary and metastatic tumors. Left panel: Spearman correlation coefficients for tumors from Pt1 and Pt2 compared with the published rank order of PDAssigner genes for classical (y-axis) and quasimesenchymal (x-axis) tumors. Right panel: scores from PurIST subtyping for each tumor. PDAC, pancreatic ductal adenocarcinoma.

To further distinguish ductal PDAC from squamoid PDAC, we classified the subtype (classical or quasimesenchymal/basal of each tumor using RNASeq and a previously published gene panel [PDAssigner[18]]) and the PurIST technique[33] that normalizes differences in tumor cellularity (Fig 2C). Both tumors from Pt1 aligned well with the classical subtype, whereas the tumors from Pt2 were more quasimesenchymal and/or basal with the metastasis scoring more basal than the primary tumor.

Leukocyte Types Associated with Immunity in Tumors From Patient 1

We used a multiplex immunohistochemistry (mIHC) workflow[34] to quantify the densities of tumor cells, fibroblasts, and 10 leukocyte subsets (Appendix Table A1). We compared the primary tumors from both patients with the metastasis from Pt1, but limited material prevented us from analyzing the metastasis from Pt2. We selected regions of interest (ROIs) within each section that contained both KRT+ epithelial or tumor cells and CD45+ immune cells (Appendix Fig A1), followed by quantitative assessment of each ROI. The metastasis from Pt1 had more epithelial or tumor cells than her primary tumor and fewer fibroblasts than the primary tumor from Pt2 (Fig 3A). A detailed analysis of leukocyte subsets revealed that CD8+ T cells and CD4+ T cells were significantly denser in the metastasis from Pt1 compared with the primary tumor from Pt2 (Fig 3B). Additionally, the primary tumor from Pt1 had a lower density of granulocytes and a higher density of Th1-like macrophages compared with the primary tumor from Pt2 (Fig 3B). Among subsets of CD4+ T cells, we found more pro-inflammatory Th1 T cells in the metastasis from Pt1 than either primary tumor (Fig 3C). Neither Th2 nor Treg cells were significantly different among the tumors. Within CD4+ T cells, we found a greater density of both cytolytic granzyme B+ (GRZB) cells and proliferative Ki67+ cells in the metastasis from Pt1 than the primary tumor from Pt2 and a higher density of proliferative Ki67+ cells in Pt1's primary tumor compared with Pt2's primary tumor (Fig 3D). Consistent with the abundant Th1-like macrophages in Pt1's primary tumor, we found that the ratio of Th1-like to Th2-like macrophages was significantly greater in Pt1's primary tumor relative to the other 2 tumors (Fig 3E). A high granulocyte/neutrophil to lymphocyte ratio is associated with poor outcomes.[35,36] Consistent with this, the primary tumor from Pt2 had a significantly higher granulocyte to CD8+ T cell ratio than either tumor from Pt1 (Fig 3E). In addition, across ROIs, there was no correlation between the densities of CD8+ T cells and granulocytes in the tumors from Pt1, but there was a significant positive correlation in Pt2's primary tumor (P < .03, Fig 4A). Furthermore, the abundant granulocytes in Pt2's tumor were proximal to tumor cells and appeared within lumens (Fig 4B) where they may contribute to pathogenesis by occluding ducts.[37]

TABLE A1.

Antibodies and Targets Used in mIHC to Identify Phenotypes and Functions of Leukocytes

FIG A1.

(A) KRT (magenta) and CD45 (teal) expression and selected ROIs (yellow rectangles) used for mIHC analyses of tumors (scale bars represent 5 mm). Examples of tertiary lymph structures (TLSs) are indicated with white arrows. ROIs, regions of interest.

FIG 3.

(A) Mean cell density per ROI for mutually exclusive populations of Ki67+ tumor or epithelial cells, Ki67neg tumor or epithelial cells, fibroblasts, and leukocytes. (B) Mean cell density per ROI of selected leukocyte types. (C) Mean cell density per ROI of mutually exclusive CD4+ T cell subsets. (D) Densities of CD8+GRZB+ and CD8+Ki67+ T cells. (E) Relative densities of Th1-like macrophages to Th2-like macrophages and CD8+ T cells to granulocytes. For D and E, each dot represents one ROI and column heights represent the mean of ROIs. All statistical comparisons between each of the three tumors for all cell types were tested by Kruskal-Wallis one-way ANOVA. Only P values < 0.05 are given. ANOVA, analysis of variance; ROI, region of interest.

FIG 4.

(A) Correlation between the densities of CD8+ T cells and granulocytes. Each dot represents one ROI. R2 and P values are based on a simple linear regression. (B) Granulocytes (CD66b+), tumor cells (PanKRT+), and fibroblasts (ACTA2+) in primary tumors from each patient. Scale bars represent 250 μm. ROI, region of interest.

We also observed tertiary lymph structures (TLSs) at the border of both primary tumors, but these were more prevalent in the primary tumor from Pt1 (Appendix Fig A1). TLSs from Pt1 also contained significantly more B cells, CD4+ T cells, and CD8+ T cells (Appendix Fig A2) as previously reported for patients with relatively positive outcomes.[24,38]

FIG A2.

(A) Representative images of lymphocytes within TLSs. Scale bars represent 250 μm. (B) Mean number of B cells, CD4+ T cells, and CD8+ T cells within ROIs containing TLSs. P values are derived from a 2-tailed t test. ROIs, regions of interest; TLS, tertiary lymph structure.

Tissue Acquisition and Patient Consent

Human tissues were obtained with informed consent in accordance with the Declaration of Helsinki and were acquired through the Oregon Pancreas Tissue Registry under Oregon Health & Science University IRB protocol #3609.

DISCUSSION

We investigated multiple modalities and tumor characteristics of PDAC between two related patients. We found several similarities between both patients' cancers, including genes commonly altered in aggressive PDAC (KRAS, TP53, and SMAD4[39,40]) and a shared PRSS1 A16V germline mutation that may predispose to pancreatitis[41-44] but is variably penetrant,[45] raising the possibility that this specific alteration leads to subclinical pancreatitis that accelerates tumorigenesis, consistent with Pt2's early onset of disease.[41,46]

The disease course for Pt1 was exceptional in the context of occult, chemotherapy-resistant metastatic disease for nearly 4 years. We identified several differences between Pt1 and Pt2 that may alone—or in combination—account for the divergent outcomes. An FANCA mutation in the metastasis from Pt1 might have sensitized that tumor to adjuvant gemcitabine and Xeloda and later to SBRT; however, Pt1's lung metastasis progressed during adjuvant chemotherapy, and no adjuvant treatment was given after surgical resection of the lung metastasis, suggesting that the indolent disease might have also been controlled by other factors. Both tumors from Pt2 contained an alteration in CDKN2A, and the metastasis from Pt2 had a copy number loss of PTEN; alterations in these genes are associated with poor outcomes and drug resistance.[47-49]

Pt1's classical subtype tumors and metastases limited to the lungs are both favorable prognostic factors. Patients with classical subtype tumors survive longer (25-30 months) than patients with basal-type tumors (10-15 months)[17-19] and are less likely to have recurrent, metastatic disease.[14,15] Patients with metastases restricted to the lungs survive on average 23 months from diagnosis[8]; Pt1 has survived > 46 months with asymptomatic metastatic disease.

Our data link the indolent disease in Pt1 with reported indicators of tumor immunity. Specifically, more prevalent TLSs at the periphery of the primary tumor (Appendix Fig A1) and denser CD8+ T cells (Fig 3B) that did not correlate with the density of granulocytes[24,50] (Fig 4A), opening the possibility that tumor cells and/or CD4+ T cells in Pt1's tumors recruit fundamentally different leukocyte cell types from Pt2's tumors.[51] Additionally, granulocytes typically promote immunosuppression in late-stage tumors,[52] consistent with the relatively low number of T cells in Pt2's tumor.

Classical subtype PDAC with lung metastases and unconventional tumor immunity may lead to exceptional outcomes. Genetic analyses and tumor leukocyte phenotyping of larger cohorts of patients with PDAC lung metastases may reveal whether metastatic organotropism is modulated by tumor immunity and/or PDAC subtype.

METHODS

See Appendix 1, Supplemental Material.

TABLE A2.

Target Combinations Used to Define the Leukocyte Types Reported