Introduction

Invasive lobular breast carcinoma (ILC) is the second most common histological breast carcinoma (BC) subtype [1-3]. ILC accounts for approximately 15% of all BC cases and is defined by distinct histomorphological characteristics reviewed previously [2].

ILC is a special type of BC, in terms of both tumor biology and clinical behavior [4, 5]. ILC is hormone receptor (HR)-positive/HER2-negative (i.e. luminal) in the vast majority of cases and is mainly driven by inactivation of the CDH1/E-cadherin cell adhesion molecule [6-9]. ILC shows a distinct landscape of mutational alterations (e.g. different frequencies of mutations in ARID1A, CDH1, ERBB2, GATA3, TP53, and other cancer-related genes) [8, 10]. In addition, ILC is over-represented in metastatic BC and is associated with distinct metastatic sites [11, 12]. The prognostic impact of molecular profiling assays, such as the Oncotype DX RS score and the PAM50 ROR score, which aid in clinical decisions for or against adjuvant chemotherapy, differs between ILC and non-lobular BC [13-16]. Regarding Oncotype DX, ILC is associated with a three-fold lower prevalence of high-risk RS scores, but the 5-year disease-free survival is similar in patients with lobular and non-lobular BC [13, 15]. Regarding PAM50, the 10-year distant recurrence rates for patients with intermediate risk ROR scores are nearly twice as high for ILC as for BC of no special type (NST, 18 versus 10%) [16]. Hence, correct classification of BC as ILC is thought to be prerequisite for adequate interpretation of prognostic profiling assays [14-16]. Moreover, correct classification of ILC is relevant for MRI indication [17, 18].

Recently, exploratory subgroup analyses from two large clinical BC trials have indicated suboptimal accuracy of the histopathological diagnosis of ILC. In the MINDACT trial, central histology review confirmed ILC in only 60% (395/654) of BC cases classified as ILC by local assessment (Cohen's κ approximately 0.68) [19]. In the WSG PlanB trial, central histology review confirmed ILC in 66% (253/385) of BC cases classified as ILC by local assessment (Cohen's κ = 0.70) [15]. However, assessment of BC subtypes was not the primary study aim of these clinical trials.

Few morphological studies have ever determined the agreement among pathologists for the diagnosis of ILC [20-22]. These studies were conducted up to 30 years ago and did not include ancillary immunohistochemistry (IHC) for E-cadherin [20-22]. The aim of the present study was to assess the degree of agreement among pathologists for the diagnosis of ILC (with and without E-cadherin IHC). In addition, this study sought to identify potential sources of discordant subtype calls.

Materials and methods Tumor specimens

Tumor specimens included 123 formalin-fixed paraffin-embedded (FFPE) core needle biopsies (CNBs) with invasive, HR-positive/HER2-negative early BC (Table 1). Tumor specimens were from patients enrolled in the West German Study Group (WSG) ADAPT trial (NCT01779206) [23, 24]. Eighty-one BCs of NST (according to central pathology review within the ADAPT trial) and 42 ILCs (according to central pathology review within ADAPT) were randomly selected from the ADAPT Trial Translational Research Registry (German Cancer Aid, grant 70112954) based on study IDs. Tumor characteristics are provided in Table 1 and in supplementary material, Tables S1–S4. FFPE tissue blocks and histological sections, which had originally been prepared for central pathology review in the ADAPT trial, were retrieved from the central tumor bank at the Hannover Medical School. Next, BCs of NST were spiked with ILCs at a ratio of approximately 2:1. Case order was arbitrary. All specimens were anonymized. This study was approved by the local ethics committee (MHH, Hannover, Germany, reference number 2716-2015).

Table 1. Tumor characteristics, as defined by the reference standard, are balanced between set A and set B. All cases Set A Set B Reference n % n % n % Test P value All cases 123 100 61 50 62 50 Subtype NST 81 100 40 49 41 51 FET 0.948 ILC 42 100 21 50 21 50 Grade G1 3 100 1 33 2 67 CSTT 0.316 G2 79 100 43 54 36 46 G3 41 100 17 41 24 59 mBSR: architecture 1 0 100 0 0 0 0 CSTT 0.325 2 22 100 13 59 9 41 3 101 100 48 48 53 52 mBSR: nuc. grade 1 7 100 3 43 4 57 CSTT 0.786 2 73 100 38 52 35 48 3 43 100 20 47 23 53 mBSR: proliferation 1 22 100 13 59 9 41 CSTT 0.239 2 77 100 38 49 39 51 3 24 100 10 42 14 58 ER Neg. 1 100 1 100 0 0 FET 0.492 Pos. 121 100 59 49 62 51 N.A. 1 100 1 100 0 0 PR Neg. 9 100 5 56 4 44 FET 0.743 Pos. 114 100 56 49 58 51 HER2 0/1+ 101 100 48 48 53 52 CSTT 0.247 2+/F.-neg. 20 100 13 65 7 35 2+/F. N.A. 1 100 0 0 1 100 3+, 2+/F.-pos.* 1 100 0 0 1 0 Ki67 <10% 6 100 3 50 3 50 CSTT 0.929 10–19% 37 100 17 46 20 54 20–34% 70 100 37 53 33 47 35–100% 10 100 4 40 6 60 E-cadherin Neg. 41 100 20 49 21 51 FET 1.000 Aberrant 0 100 0 0 0 0 Pos. 82 100 41 50 41 50 CDH1 status Wild-type 90 100 45 50 45 50 FET 1.000 Mutant 33 100 16 48 17 52 Beta-catenin Neg. 37 100 18 49 19 51 CSTT 0.950 Focally pos. 4 100 2 50 2 50 Pos. 82 100 41 50 41 50 p120-catenin Neg. 12 100 7 58 5 42 FET 0.559 Pos. 111 100 54 49 57 51 p120-catenin mislocation Membranous 76 100 40 53 36 47 FET 0.228 Mislocated 35 100 14 40 21 60 Not informative 12 100 7 58 5 42 P-cadherin Neg. 102 100 53 52 49 48 FET 0.338 Focally pos. 21 100 8 38 13 62 Pos. 0 100 0 0 0 0 CSTT, chi-square test for trends (set A versus set B); ER, estrogen receptor; F., HER2 fluorescence in situ hybridization (FISH); FET, Fisher's exact test (set A versus set B); mBSR, modified Bloom–Scarf–Richardson Score grading components; neg., negative; pos., positive; PR, progesterone receptor. * Corresponds to one case with a minor HER2-pos. Subclone, <5% of cells. Reference diagnosis

Specimens were pre-annotated with (1) histological BC subtype according to local pathologies within the ADAPT trial (documented in 2012–2016); (2) histological BC subtype according to central pathology review within the ADAPT trial (diagnosis made by a team of 2–4 expert breast pathologists headed by Prof. HK, based on hematoxylin/eosin [HE]-stained sections, aided by E-cadherin IHC [clone ECH-6, Zytomed, Berlin, Germany] for all cases, documented in 2012–2016); (3) additional characteristics including histological grade (modified Bloom–Scarff–Richardson score), grading score components, histological variants (when applicable), estrogen receptor, progesterone receptor, and HER2 status, Ki67 index, and E-cadherin status (according to central IHC and central review within ADAPT, documented in 2012–2016); (4) molecular features including beta-catenin, p120-catenin, and P-cadherin expression (central IHC, documented in 2018–2019); and (5) CDH1/E-cadherin mutation status (according to central next-generation sequencing (NGS), documented in 2018–2020). Tumor characteristics according to central pathology review within the ADAPT trial served as a pre-defined reference standard in this study.

Immunohistochemistry

IHC was carried out in the central pathology unit of the ADAPT trial, using a Benchmark Ultra automated stainer (Ventana, Tucson, AZ, USA). Central IHC scoring was carried out by a team of 2–4 expert pathologists on a multi-headed microscope. Immunological reagents and IHC scoring methods are summarized in the supplementary material, Table S1.

Next-generation sequencing

Genomic DNA was extracted as described previously [25]. The CDH1/E-cadherin mutation status was determined by NGS using a customized CDH1 NGS panel and the Ion S5 system (Life Technologies, Carlsbad, CA, USA). This NGS panel covered the complete protein-coding sequence of the CDH1 gene, the 5′-untranslated region (UTR) sequence of exon 1, and the 3′-UTR sequence of exon 16, as described previously [25, 26]. Variant annotation was performed with ANNOVAR software and database tools (http://www.openbioinformatics.org/annovar) [27]. Due to limited tumor tissue in CNBs, matched FFPE resection specimens corresponding to the CNBs were used for mutational analysis.

Slide sets

For virtual microscopy, histological sections were scanned using a dotSlide scanner microscope (Olympus GmbH, Münster, Germany). Slides were divided into two sets for re-assessment based on HE-stained sections (set A, n = 61 cases) and for re-assessment based on HE-stained sections and E-cadherin IHC (set B, n = 62 cases). Tumor characteristics were balanced (Table 1).

Participants

Participants included 35 experienced board-certified pathologists from 27 institutions from nine countries (Belgium, France, Germany, Hungary, Ireland, Italy, The Netherlands, Portugal, and Switzerland). The approximate geographical distribution of participants is illustrated in supplementary material, Figure S1. Of the 35 pathologists, 28 (80%) were from academic institutions or university clinics and had special interest in BC. Of the 35 participants, 7 (20%) were general pathologists from non-academic institutions, and all were involved in BC diagnostics on a regular basis. Three pathologists who had been involved in the ADAPT trial central review (in 2012–2016) participated in the re-assessment in 2019–2020 (participant IDs p01_i01, p02_i01, and p03_i01). For histological re-assessment, all participants independently interpreted all cases from slide sets A and B. In 2019, participants could opt for glass slides or virtual microscopy. In 2020–2021, participants could only opt for virtual microscopy, which was related to restrictions during the coronavirus pandemic. Using checkmark matrices, participants classified each specimen as BC of NST/non-lobular BC versus mixed BC (NST or other non-lobular BCs mixed with an ILC component) versus ILC. In set B, ancillary E-cadherin IHC stainings were classified as positive versus aberrant (such as nuclear mislocalization or fragmented staining) versus negative. Participants were instructed to make their classification calls as they would usually do in routine diagnostics. Written text comments were optional. On the discretion of individual participants, strongly reduced E-cadherin immunoreactivity was occasionally classified as an aberrant E-cadherin status, and written text comments were provided in such instances. All participants were blinded to the reference standard and NGS results.

Statistics

For two-dimensional presentation of BC subtype calls, specimens and participants were clustered using single linkage and ClustVis software [28]. For assessment of inter-observer agreement, pairwise Cohen's κ values (ranging from −1 to 1) for the exact BC subtype (NST/non-lobular BC versus mixed BC versus ILC) were calculated for each pair of pathologists (n = 595 pairs) using JMP software (JMP 11, SAS Institute Corporation, Cary, NC, USA). For assessment of agreement with the reference standard, Cohen's κ values were also calculated based on each participant's subtype calls and the reference standard using VassarStats [29]. Interquartile range (IQR) was calculated with GraphPad Prism 5 (GraphPad Software, Inc., San Diego, CA, USA). Cohen's κ values were interpreted as follows: <0.0 (poor agreement), 0.0–0.20 (slight agreement), 0.21–0.40 (fair agreement), 0.41–0.60 (moderate agreement), 0.61–0.80 (substantial agreement), and 0.81–1.00 (almost perfect agreement) [30]. The Wilcoxon test was used to assess statistical significance of different median κ values obtained in sets A and B. Accuracy was calculated as the proportion of cases concordantly classified as NST/non-lobular BC versus mixed BC/ILC by participants and the reference (accuracy for the detection of a lobular tumor component). Statistical significance of different proportional participant calls for ILC in ILC subsets with different growth pattern (dissociated, single files, and trabecular) was determined with the Kruskal–Wallis test.

Results Baseline characteristics

Two sets of histological slides (sets A and B) were compiled from HR-positive/HER2-negative BCs. Tumors were randomly selected from patients enrolled in the WSG ADAPT trial (NCT01779206) [23, 24]. Specimens were pre-annotated with BC subtypes according to local pathologies and according to central pathology review in the ADAPT trial (based on expert assessment and aided by upfront E-cadherin IHC for all cases). BC subtypes and E-cadherin status according to central review served as a pre-defined reference standard. Tumor characteristics were balanced between sets A and set B (Table 1).

ILCs, as defined by the reference standard, associated with loss of E-cadherin expression, CDH1/E-cadherin mutation, loss of beta-catenin expression, and aberrant cytosolic/nuclear p120-catenin (all p < 0.001) (supplementary material, Table S4). In detail, loss of E-cadherin, as defined by the reference IHC status, was evident in 40/42 (95%) ILCs and in 1/80 (1%) BC of NST. The CDH1 mutation frequency was 32/42 (76%) in ILC and 1/82 (1%) in BC of NST (supplementary material, Table S4). One ILC harbored two different mutations (case B031). Most CDH1 mutations (26/34, 76%) were frameshift or nonsense mutations generating premature stop codons (supplementary material, Figure S2). E-cadherin expression was lost in 31/34 (91%) BCs harboring CDH1 mutations. Conversely, E-cadherin expression was preserved in 3/34 (9%) BCs harboring CDH1 mutations, all of which were missense mutations (supplementary material, Figure S2). The frequency of CDH1 mutations was balanced between sets A and B (Table 1). Regarding histological growth patterns, 33/42 (79%) ILCs were classic ILCs with predominant dissociated growth pattern or single file growth pattern. The remaining ILCs (9/42, 21%) showed trabecular or solid growth patterns and were balanced between sets A and B (supplementary material, Table S3). Rare ILC variants, such as histiocytoid ILC [reviewed in Ref. 2], were not included.

BC subtype calls by local pathologies in the ADAPT trial showed substantial agreement with the reference standard in set A (κ = 0.74) and in set B (κ = 0.71) (Table 2). This is consistent with previous findings in the WSG PlanB trial (κ = 0.70) [15].

Table 2. Agreement between local and central pathology (in the ADAPT trial).