Heterogeneity of small duct‐ and large duct‐type intrahepatic cholangiocarcinoma

The histological subtype of intrahepatic cholangiocarcinoma (iCCA) is associated with different mutational characteristics that impact clinical management. So far, data are lacking on the presence of small duct iCCA (SD‐iCCA) and large duct iCCA (LD‐iCCA) in a single patient. The aim of the current study was to determine the presence and degree of intratumoural heterogeneity of SD‐ and LD‐iCCA features in different tumour regions.


Heterogeneity of small duct-and large duct-type intrahepatic cholangiocarcinoma
Aims: The histological subtype of intrahepatic cholangiocarcinoma (iCCA) is associated with different mutational characteristics that impact clinical management.So far, data are lacking on the presence of small duct iCCA (SD-iCCA) and large duct iCCA (LD-iCCA) in a single patient.The aim of the current study was to determine the presence and degree of intratumoural heterogeneity of SD-and LD-iCCA features in different tumour regions.Methods and results: All patients treated with surgically resected iCCA at Frankfurt University Hospital between December 2005 and March 2023 were retrospectively analysed.Histomorphological features of SD-and LD-iCCA were evaluated by an expert hepatobiliary pathologist.Tissue samples suspicious for subtype heterogeneity were further investigated.Immunohistochemistry for N-cadherin, S100P, MUC5AC, MUC6, TFF1 and AGR2 and mutational profiling with the Illumina TruSight Oncology 500 (TSO500) assay were performed separately for the SD-and LD-iCCA regions.Of 129 patients with surgically resected iCCA, features of either SD-or LD-iCCA were present in 67.4% (n = 87) and 24.8% of the patients (n = 32), respectively; 7.8% (n = 10) had histomorphological features of both SD-and LD-iCCA, seven patients (5.4%) of which had sufficient formalin-fixed, paraffin-embedded tissue for further analysis.Heterogeneity of both subtypes could be confirmed with immunohistochemistry.In five of seven (71.4%) patients, molecular profiling revealed intratumoural differences in genetic alterations between the SD-and LD-iCCA region.In one patient, a BRAF mutation (p.V600E) was found in the SD-iCCA but not in the LD-iCCA region of the tumour.Conclusions: A marked portion of patients with iCCA exhibits both SD-and LD-iCCA in different tumour

Introduction
2][3] On one hand, small duct iCCA (SD-iCCA) was found to be more peripheral in the liver, resembling a ductular and cholangiolar type with no or only limited mucin production. 4][11][12] Against this background, the aim of the current study was to retrospectively determine the presence and degree of intratumoural subtype-heterogeneity of our iCCA cohort on a histological as well as mutational level.

Methods
All patients treated with surgically resected (R0, R1) iCCA at Frankfurt University Hospital between December 2005 and March 2023 were retrospectively analysed.Patients with clear histomorphological features of the same subtype throughout the tumour tissue were excluded from further investigation.Tissue samples with histomorphological features of both SD-iCCA and LD-iCCA in different areas of the same tumour were further evaluated.Representative images in haematoxylin and eosin (H&E) staining of all enrolled cases harbouring histomorphological features of both SD-iCCA and LD-iCCA are shown in Figure 1.Representative macroscopic images and the corresponding H&E stains of the different tumour features on separate capsules from the same patient are depicted in Supporting information, Figure S1.Immunohistochemistry (IHC) of both SD-iCCA and LD-iCCA regions for each patient was performed for N-cadherin, S100P, MUC5AC, MUC6, trefoil factor 1 (TFF1) and anterior gradient homologue 2 (AGR2) after histomorphological characterisation (Supporting information, Figure S2).Full information on patient selection, IHC and next-generation sequencing is provided in the Supporting information.

P A T I E N T S A N D C L I N I C A L C H A R A C T E R I S T I C S
Of the 129 patients with iCCA, clear histomorphological features of either SD-or LD-iCCA were present in 67.4% (n = 87) and 24.8% of the patients (n = 32), respectively; 7.8% (n = 10) had histomorphological features of both SD-and LD-iCCA in different regions of the tumour, while seven patients (5.4%) with sufficient FFPE tissue (male, n = 5; mean age = 62.3, range = 46-72 years) were included in our study for further analysis (Supporting information, Figure S3).Clinical characteristics are shown in Supporting information, Table S1.
Four of seven patients were predominantly SD-iCCA harbouring a minor LD-iCCA component, while three of seven patients were predominantly LD-iCCA with a minor SD-iCCA component (Table 1).The presence of either the SD-or LD-component in recurrent and metastatic disease are depicted in Supporting information, Table S2.Comparison of IHC between the SD-iCCA and LD-iCCA region for the enrolled patients also revealed an intratumoural heterogeneity.In line with the literature, N-cadherin was positive in 86% (n = 6) of the SD-iCCA regions, whereas it was negative in the LD-iCCA counterparts (Supporting information, Table S3).Next, the examination of S100P, MUC5AC, MUC6 and TFF1 revealed a slight and mainly quantitative up-regulation in LD-iCCA regions Intrahepatic cholangiocarcinoma 1063 compared to the SD-iCCA counterparts in our patients.
All seven patients with intratumoural heterogeneity of SD and LD-iCCA received separate molecular profiling of both regions using the TSO500 assay.In total, 54 pathogenic or probably pathogenic variants were found in 21 altered genes.In 11 of 14 samples (78.6%), a pathogenic or probably pathogenic variant was detected, whereas no genetic alteration was found in only both the small duct-and the large duct-type regions of patient 4 (Table 1).Interestingly, molecular profiling revealed intratumoural differences of genetic alterations in five of seven patients (71.4%) between the SD-and LD-iCCA regions, while five of seven patients also shared common mutations (Table 1).For example, both the small duct-and the Only pathogenic or probably pathogenic alterations are reported.Altered genes are reported multiple times per sample when different variants are affected.The percentage (%) of the SD-and LD-iCCA component for each patient is based on the pathologist's assessment.ACMG, American College of Medical Genetics and Genomics; LD-iCCA, large duct-type; SD-iCCA, small duct-type.

I M P A C T O F I N T R A T U M O U R A L H E T E R O G E N E I T Y O N T H E R A P E U T I C O P T I O N S
All genetic variants with a CVI score ≥ 6 between the SD-and LD-iCCA region for each patient were compared.Therapeutically relevant alterations could be detected in 57.1% of the samples (n = 8), whereas no genetic alteration with a CVI score ≥ 6 was found only in both the small duct-and the large duct-type regions of patient 6. Remarkably, in four of seven patients (57.1%) patients, CVI scores revealed intratumoural differences in therapeutically relevant alterations between the SD-and LD-iCCA regions (Supporting information, Table S5).For example, the BRAF mutation (p.V600E) in the SD-iCCA region of patient 7 provides 12 drug options that are lacking in the LD-iCCA region of the tumour.A detailed overview of the genetic alterations and the corresponding drug options is provided in Supporting information, Table S6.There was no impact upon overall survival between clear SD, LD and mixed SD/LD iCCA (data not shown).

Discussion
Determination of the histological subtype of iCCA was recently implemented in routine pathological workup, as it is associated with different mutational characteristics with impact upon the clinical management.The aim of the present study was to determine the presence and degree of intratumoural heterogeneity of patients harbouring features of both SD-iCCA and LD-iCCA.To our knowledge, this study is the first to investigate this clinically relevant topic.
In the present study, 67% of the patients had SD-iCCA, while 25% were suffering from LD-iCCA and 8% had features of both types.8]13 In line with our data, an indeterminate type was described in 8-13%, although whether or not these cases had features of both subtypes is not described in detail. 6,7While data from other studies on histological subtype heterogeneity are missing, several recent studies observed a relevant degree of intratumoural heterogeneity on a mutational level.For example, Chen et al. showed in a multiomics analysis on 16 patients that the proportion of truncal mutations varies between 24% in patients with high heterogeneity and 68% in patients with low heterogeneity. 14Similarly, in a study from 2022 on 207 samples of 45 patients, Lin et al. could demonstrate that 54% of the driver mutations were subclonal. 9While FGFR2 fusions were all clonal, in some patients therapeutically relevant IDH1/2 mutations were found to be subclonal.These findings correspond with the study of Xiang et al., who found a significant proportion of mutations in IDH1/2 to be non-truncal, indicating that this mutation might be a subclonal driver. 11As IDH1 mutations typically occur only in SD-iCCA, but not in LD-iCCA, these findings highlight the potential relevance of subtype heterogeneity for clinical management and strengthen the need to considerate obtaining a new biopsy in case of recurrence.If this is not possible, diligent investigation of the initially resected surgical specimen by an expert hepatobiliary pathologist is of high importance to detect potential presence of subtype heterogeneity.As shown in the present study, the distinction between the two components should be based primarily upon histomorphology, although immunohistochemistry may be helpful to further characterise SD-iCCA and LD-iCCA if subtype heterogeneity is suspected.Furthermore, if there is evidence for subtype heterogeneity, mutational profiling of the respective regions might be considered.
To strengthen our findings, we performed a comprehensive molecular analysis of both suspected SD-iCCA and LD-iCCA subtype regions.As five of seven patients also shared common mutations, it can be assumed that both regions derive from the same clone.However, in line with the studies mentioned above, we also observed different therapeutic genetic alterations in five of seven patients.Moreover, in one patient a therapeutically relevant BRAF mutation was found only in the part with SD-iCCA, but not in the large duct counterpart.This patient demonstrates that the detection of subtype heterogeneity with consecutive mutational profiling can lead to the identification of currently available targeted therapeutical options being recommended as second-line treatment by international treatment guidelines. 15,16As the majority of iCCA patients is irresectable when diagnosed, future studies evaluating the intratumoural heterogeneity based upon multiple biopsies from different tumour regions are warranted.Notably, histological subtype heterogeneity may also be of particular interest in the adjuvant setting, as in these cases complete surgical specimens are available and directed therapies are currently under investigation in clinical studies. 17n conclusion, our data strengthen the high relevance of determining the subtype of every patient with iCCA.In the case of presence of histopathological heterogeneity, mutational profiling of multiple samples should be taken into consideration to reveal potential targetable therapeutic options.

Figure 1 .
Figure 1.Representative images of histomorphological features of small duct-and large duct-type iCCA in different regions of the tumour.Representative histomorphological features of small duct-and large duct-type iCCA in haematoxylin and eosin stain.Scale bars = 100 lm.Ó 2024 The Authors.Histopathology published by John Wiley & Sons Ltd., Histopathology, 84, 1061-1067.

Table 1 .
Molecular alterations of small duct-and large duct regions in iCCA