Clinical and molecular characterization of HER2 amplified-pancreatic cancer

  • Angela Chou1, 2, 3Email author,

    Affiliated with

    • Nicola Waddell6Email author,

      Affiliated with

      • Mark J Cowley1, 3Email author,

        Affiliated with

        • Anthony J Gill1, 4, 5,

          Affiliated with

          • David K Chang1, 12, 13,

            Affiliated with

            • Ann-Marie Patch6,

              Affiliated with

              • Katia Nones6,

                Affiliated with

                • Jianmin Wu1, 3,

                  Affiliated with

                  • Mark Pinese1, 3,

                    Affiliated with

                    • Amber L Johns1,

                      Affiliated with

                      • David K Miller6,

                        Affiliated with

                        • Karin S Kassahn6,

                          Affiliated with

                          • Adnan M Nagrial1, 3,

                            Affiliated with

                            • Harpreet Wasan7,

                              Affiliated with

                              • David Goldstein8,

                                Affiliated with

                                • Christopher W Toon4, 5, 9,

                                  Affiliated with

                                  • Venessa Chin1, 3,

                                    Affiliated with

                                    • Lorraine Chantrill1, 3, 10,

                                      Affiliated with

                                      • Jeremy Humphris1,

                                        Affiliated with

                                        • R Scott Mead1, 2, 3,

                                          Affiliated with

                                          • Ilse Rooman1, 3,

                                            Affiliated with

                                            • Jaswinder S Samra11,

                                              Affiliated with

                                              • Marina Pajic1, 3,

                                                Affiliated with

                                                • Elizabeth A Musgrove1, 12,

                                                  Affiliated with

                                                  • John V Pearson6,

                                                    Affiliated with

                                                    • Adrienne L Morey2Email author,

                                                      Affiliated with

                                                      • Sean M Grimmond6, 12Email author and

                                                        Affiliated with

                                                        • Andrew V Biankin1, 12, 13Email author

                                                          Affiliated with

                                                          Genome Medicine20135:78

                                                          DOI: 10.1186/gm482

                                                          Received: 11 June 2013

                                                          Accepted: 23 August 2013

                                                          Published: 31 August 2013

                                                          Abstract

                                                          Background

                                                          Pancreatic cancer is one of the most lethal and molecularly diverse malignancies. Repurposing of therapeutics that target specific molecular mechanisms in different disease types offers potential for rapid improvements in outcome. Although HER2 amplification occurs in pancreatic cancer, it is inadequately characterized to exploit the potential of anti-HER2 therapies.

                                                          Methods

                                                          HER2 amplification was detected and further analyzed using multiple genomic sequencing approaches. Standardized reference laboratory assays defined HER2 amplification in a large cohort of patients (n = 469) with pancreatic ductal adenocarcinoma (PDAC).

                                                          Results

                                                          An amplified inversion event (1 MB) was identified at the HER2 locus in a patient with PDAC. Using standardized laboratory assays, we established diagnostic criteria for HER2 amplification in PDAC, and observed a prevalence of 2%. Clinically, HER2- amplified PDAC was characterized by a lack of liver metastases, and a preponderance of lung and brain metastases. Excluding breast and gastric cancer, the incidence of HER2-amplified cancers in the USA is >22,000 per annum.

                                                          Conclusions

                                                          HER2 amplification occurs in 2% of PDAC, and has distinct features with implications for clinical practice. The molecular heterogeneity of PDAC implies that even an incidence of 2% represents an attractive target for anti-HER2 therapies, as options for PDAC are limited. Recruiting patients based on HER2 amplification, rather than organ of origin, could make trials of anti-HER2 therapies feasible in less common cancer types.

                                                          Background

                                                          Pancreatic cancer is the fourth leading cause of cancer death in western societies, with a 5-year survival rate of less than 5% [1]. Systemic therapies are only modestly effective; however, there is emerging evidence that small groups of patients may respond well to specific treatments [2, 3]. Current therapeutic development is focused on targeting molecular mechanisms, and this has resulted in significant improvements in outcome for several cancer types (for example, crizotinib for EML4-ALK fusion-positive non-small cell lung cancer (NSCLC)). This approach is shifting the traditional organ-based classification of cancer towards a new molecular taxonomy, and creating opportunities to apply therapeutics for the treatment of cancers originating in other organs that harbour similar molecular anomalies. Such indications for extension of existing therapeutics is attractive; however, specific molecular phenotypes and diagnostic characteristics are complex and usually inadequately defined [4, 5]. The target population for cancers of organs, apart from where the therapeutic strategy was initially developed, often occur at low frequency, further adding to the challenge.

                                                          Emerging data from cancer sequencing initiatives such as the International Cancer Genome Consortium (ICGC) [6] and The Cancer Genome Atlas (TCGA) [7] are unveiling a vast heterogeneity of molecular aberrations in cancer. Pancreatic ductal adenocarcinoma (PDAC), the predominant form of pancreatic cancer, is particularly heterogeneous, and apart from a few notable exceptions, which have not been successfully targeted, most genetic aberrations have a frequency of 2% or less [810].

                                                          Trastuzumab, a monoclonal antibody that targets the HER2 receptor, is an effective therapy for HER2-amplified breast cancer, and was recently extended to the treatment of HER2-amplified gastric cancer [11]. In addition, borderline signals have been seen in clinical trials of semi-selected patients with NSCLC [4], and case reports describe exceptional responses to trastuzumab in other cancers with HER2 amplification, such as cholangiocarcinoma [12]. Defining HER2 amplification as a biomarker of trastuzumab responsiveness is integral to targeting appropriate populations for therapy. Although HER2 overexpression and amplification has been assessed in PDAC (see Additional file 1), standardized diagnostic assays have on the whole not been applied, and the roles of emerging diagnostic approaches such as genomic sequencing are yet to be explored. As a consequence, the diagnostic criteria and prevalence of HER2 amplification in PDAC remain unclear. Although preclinical studies support potential efficacy of trastuzumab in PDAC [13, 14], clinical trials have been hampered by non-standardized assays and a consequent lack of focus on appropriate subgroups [15, 16]. With the current poor survival, low therapeutic responsiveness, and vast molecular heterogeneity of PDAC, even if a relatively low proportion were found to be HER2-amplified, targeting by HER2 amplification represents an attractive potential therapy.

                                                          In this study, we identified HER2 amplification in diagnostic specimens of PDAC using single nucleotide polymorphism (SNP) arrays and whole genome sequencing. We defined the characteristics and prevalence (2.1%) of HER2 amplification in a large cohort of patients with resected PDAC using standardized reference laboratory assays. We found that HER2-amplified PDAC has an atypical pattern of metastatic spread with a predilection for lung metastasis and local recurrence, rather than liver metastases. Assessment of HER2 amplification across 16 cancer types suggested a prevalence of at least 22,000 cases per annum in the USA (excluding breast and gastric cancer), suggesting that a molecular recruitment strategy may make it feasible to test anti-HER2 therapies in less common cancer types.

                                                          Methods

                                                          Ethics approval

                                                          Ethics approval for acquisition of data and biological material was obtained from the human research ethics committee at each participating institution, conducted in accordance with the National Statement on Ethical Conduct in Human Research 2007 and the Declaration of Helsinki. Consent was obtained from prospectively recruited patients for genomic sequencing through the Australian Pancreatic Cancer Genome Initiative (APGI) as part of the ICGC. Consent was waived by Human Research Ethics Committees for retrospectively acquired data and material under an approved protocol (see Additional file 2).

                                                          Genomic sequencing, copy number, and mRNA expression analysis

                                                          Patients were prospectively recruited to the APGI [17] for genomic sequencing as part of the ICGC, and details of sample acquisition and processing have been described previously [9]. Briefly, samples focused on primary operable non-pretreated PDAC. Tissue was prepared by either full face frozen sectioning or the ends being excised and processed in formalin, then representative sections were reviewed by at least one pathologist to verify presence of carcinoma in the sample to be sequenced, and to estimate the percentage of malignant epithelial nuclei in the sample relative to stromal nuclei. Nucleic acids were extracted from fresh frozen tumour and normal tissue pairs, and whole genome and exome sequencing was performed using a combination of long mate pair and paired-end approaches. DNA copy number was assessed using specific microarrays (HumanOmni1-Quad BeadChip, Illumina Inc., San Diego, CA, USA). Primary tumour mRNA expression was assayed using human microarrays (HT-12 V4; Illumina) (GEO accession GSE36924). Gene expression profiles were classified into intrinsic breast cancer subtypes using the PAM50 classifier [18].

                                                          Defining prevalence and diagnostic criteria for HER2 amplification

                                                          Formalin-fixed, paraffin wax-embedded diagnostic material from a cohort of 469 patients who had undergone operative resection for PDAC was accrued from 12 institutions associated with the APGI between 1990 and 2012. Subsets of this cohort have previously been published [19, 20], and detailed characteristics are presented (see Additional file 3: Table S2).

                                                          HER2 immunohistochemistry (IHC) and in situ hybridization (ISH) was performed in a national reference HER2 diagnostic testing laboratory. Scoring for HER2 protein expression and amplification was based on criteria recommended for breast and gastric carcinoma (Table 1) [21].
                                                          Table 1

                                                          IHC and FISH scoring for the detection of HER2 amplification a

                                                          Her2 IHC score

                                                          IHC scores

                                                          HER2 FISH

                                                           

                                                          (n = 469)

                                                          Positive, n = 10

                                                          Negative, n = 459

                                                          3+

                                                          7 (1.5%)

                                                          7

                                                          0

                                                          2+

                                                          27 (5.8%)

                                                          3

                                                          24

                                                          1+

                                                          59 (12.6%)

                                                          0

                                                          59

                                                          0

                                                          376 (80.2%)

                                                          0

                                                          376

                                                          Abbreviations: FISH fluorescence in situ hybridization; IHC immunohistochemistry; PDAC pancreatic ductal adenocarcinoma.

                                                          aThe criteria used for scoring HER2 by immunohistochemistry and in situ hybridization in PDAC were as follows. Her2 IHC score criteria (modified from Hofmann et al[21]): 0, no staining of any pattern or intensity; 1+. weak discernable membrane staining; 2+, mild to moderate complete or basolateral membrane staining; 3+, strong, complete, or basolateral membrane staining. HER2 ISH criteria: non-amplified, Her2 count <4 and Her2:cep17 ratio <2; amplified, Her2 count ≥4 and Her2:cep17 ratio ≥2.

                                                          Results

                                                          In the initial set of 50 patients recruited to the APGI, SNP arrays detected a case of HER2 amplification (Figure 1A), with associated high HER2 mRNA expression (see Additional file 4). HER2 amplification was verified using standardized laboratory assays currently used for breast and gastric cancer at an HER2 diagnostic reference laboratory. This case was analyzed further using whole genome sequencing, copy number analysis, and mRNA expression profiling.
                                                          http://static-content.springer.com/image/art%3A10.1186%2Fgm482/MediaObjects/13073_2013_477_Fig1_HTML.jpg
                                                          Figure 1

                                                          HER2 -amplified pancreatic ductal adenocarcinoma. (A) Single nucleotide polymorphism (SNP) array showing amplification on chromosome 17 containing the HER2 locus (boxed). (B) Inverted amplification on chromosome 17, spanning HER2 and truncating MED1 and TOP2A at either end (chr17:37,565,271-38,554,848). (C) Circos plot showing mutations and structural rearrangements of patient 9 (Table 2). Genes with non-silent substitutions are shown on the outer wheel. The SNP array data are shown on the subsequent plots (copy number predictions and B-allele frequency of probes). Structural rearrangements are indicated with lines inside the circle: deletions (green), inversions (yellow), intra-chromosomal rearrangements (blue), foldback inversions (light orange), and amplified inversions (dark orange). Genes with structural variation are shown in blue if deleted and green if interrupted.

                                                          Genomic characteristics of HER2-amplified pancreatic ductal adenocarcinoma

                                                          Whole genome and exome sequencing identified several classes of mutation including single nucleotide variants, small insertions and deletions (indels), and chromosomal rearrangements (see Additional file 5). Chromosomal rearrangements included a region of loss on chromosome 18 containing the MAPK4, DCC, SMAD4, and ELAC1 genes, and three complex amplification regions: an inverted amplification of a region of chromosome 17, spanning HER2 and truncating MED1 and TOP2A at either end (chr17:37,565,271-38,554,848) (Figure 1B), and two overlapping fold back inversion events on chromosome 9: a 1.5 Mb event within a 1.9 Mb event, disrupting UBAP2. Exome sequencing identified 23 somatic mutations affecting 21 genes, including KRAS p.G12V and TP53 p.Q317, and 2 mutations in both TOP2A and RYR2 (Figure 1C).

                                                          We performed gene set enrichment analysis using all genes affected by mutation, amplification, copy number alterations (GISTIC2.0 peaks P < 0.25), and disruptions caused by structural rearrangements (see Additional file 6). There were three strong biological themes: 1) genes from the HER2-amplicon (13 genes; P<1 × 10-7); 2) a broad cancer theme driven by KRAS, TP53, and EPHA5 (13 gene sets; P<0.001); and 3) an immune signature, driven by type I interferons, IFNA2, INFA6, INFA13 (7 gene sets; P < 0.001), which are involved in recognition of viral infection and neoplasia (see Additional file 7).

                                                          Intrinsic subtype analysis using expression microarray data from the APGI cohort (n = 90) [9] classified each PDAC sample using the PAM50 classifier, which captures five breast cancer subtypes: HER2-amplified, luminal A, luminal B, basal, and normal-like [18]. The HER2-amplified patient with PDAC identified above clustered with the HER2-amplified intrinsic subtype, with a confidence of 93%. This was driven by high expression of HER2, GRB7, and FOXA1, and low expression of KRT17 and MIA (see Additional file 8).

                                                          HER2 amplification in pancreatic ductal adenocarcinoma

                                                          In order to define the clinical and histopathological characteristics, diagnostic criteria, and prevalence of HER2 amplification in PDAC, we assessed a cohort of 469 patients with resected PDAC using IHC and in situ hybridization (ISH). IHC for Her2 protein expression showed 3+ staining in 7 cases (1.5%) (Figure 2A), 2+ staining in 27 (5.8%), 1+ in 59 (12.6%) and no staining in 376 (80.2%) (Table 1). All 7 cases that exhibited Her2 3+ staining were found to be HER2-amplified on silver ISH and fluorescence ISH (FISH) with 100% concordance between the two methods (Figure 2B,C). Only 3 of 27 cases that were Her2 2+ on IHC were found to be HER2-amplified (11%). No cases of 1+ or negative Her2 IHC were HER2-amplified, and the overall prevalence of HER2 amplification was 2.1% (10 of 469 cases). All the amplified cases showed a tendency for the HER2 signal to form clusters. One case was found to have intra-tumoral heterogeneity by IHC and ISH, with distinct HER2-positive and HER2-negative areas. As HER2 amplification was only present when Her2 IHC produced 2+ or 3+ staining, IHC screening followed by ISH in cases of 2+ and 3+ IHC staining may be an appropriate and cost-effective approach for the detection of HER2-amplified PDAC (see Additional file 9).
                                                          http://static-content.springer.com/image/art%3A10.1186%2Fgm482/MediaObjects/13073_2013_477_Fig2_HTML.jpg
                                                          Figure 2

                                                          Clinicopathological features of HER2 -amplified pancreatic ductal adenocarcinoma (PDAC). (A) Her2 immunohistochemistry showing 3+ staining, and corresponding HER2 amplification on (B) silver and (C) fluorescence in situ hybridization. Imaging results of patient 9 (Table 2) showing (D) cerebral and (E) lung metastases, and (F) absence of liver metastases.

                                                          Clinical features of HER2-amplified pancreatic ductal adenocarcinoma

                                                          Of the ten patients with HER2 amplified tumours, nine died of PDAC, and one died of respiratory failure as a result of chronic obstructive pulmonary disease with no evidence of cancer recurrence. The pattern of metastatic relapse of the HER2-amplified cases was characterized by a lack of liver metastases. None of the eight cases with a documented site of recurrence had liver metastases (one patient had recurrence, and for another patient the site of recurrence was not reported). Of these eight patients, four had lung metastases (one of whom also had brain metastases (Figure 2D-F), and two also had local recurrence, although cerebral imaging was only performed in one patient), while the remaining four patients had peritoneal and local recurrences only, although imaging of the thorax was not performed in two of these patients (Table 2). The higher prevalence of lung metastasis (in the absence of liver metastases) in HER2-amplified tumours was significantly different to that of non-HER2-amplified tumours (P = 0.0022; Table 3), as was the rate of recurrence at any site without liver metastases (P = 0.0028; Table 3). HER2 amplification was not associated with any other clinicopathological characteristics such as age (P = 0.5467), sex (P = 0.7520), tumour stage (P = 0.4495), grade (P = 0.1259), or size (P = 0.4695), or disease-specific survival (P = 0.2502; Table 3). Histopathologically, there were no specific features to distinguish HER2-amplified tumours from non-HER2-amplified tumours. All HER2-amplified cases were moderately differentiated, and most had a diffuse or at least focally macroglandular architecture.
                                                          Table 2

                                                          Clinicopathological characteristics of HER2-amplified PDAC

                                                          Patient

                                                          Her2 IHC score

                                                          HER2 ratio

                                                          HER2 count

                                                          Overall survival (months)

                                                          Cause of death

                                                          Pattern of recurrence

                                                          Comments

                                                                

                                                          Lung

                                                          Brain

                                                          Liver

                                                          Local

                                                          Peritoneal

                                                           

                                                          1a

                                                          3+

                                                          2.45

                                                          6

                                                          13.6

                                                          PDAC

                                                          -

                                                          ND

                                                          -

                                                          +

                                                          +

                                                          Laparotomy showed peritoneal and local recurrence

                                                          2

                                                          3+

                                                          7.5

                                                          13

                                                          43.5

                                                          COPD

                                                          -

                                                          ND

                                                          -

                                                          -

                                                          -

                                                          CT at 43 months showed no recurrence

                                                          3

                                                          3+

                                                          4.12

                                                          10

                                                          5.0

                                                          PDAC

                                                          -

                                                          ND

                                                          -

                                                          +

                                                          -

                                                          Local recurrence

                                                          4b

                                                          2+

                                                          2.5

                                                          5.4

                                                          36.5

                                                          PDAC

                                                          +

                                                          ND

                                                          -

                                                          +

                                                          +

                                                          Ascites/pleural effusions, no liver metastases on CT

                                                          5

                                                          3+

                                                          4.38

                                                          8.1

                                                          18.6

                                                          PDAC

                                                          ND

                                                          ND

                                                          ND

                                                          ND

                                                          ND

                                                          Site not documented

                                                          6

                                                          2

                                                          3.56

                                                          7

                                                          10.1

                                                          PDAC

                                                          ND

                                                          ND

                                                          -

                                                          +

                                                          ND

                                                          R2 resection

                                                          7

                                                          3+

                                                          2.7

                                                          4.5

                                                          41.6

                                                          PDAC

                                                          ND

                                                          ND

                                                          -

                                                          +

                                                          +

                                                          No liver metastases on CT; peritoneal recurrence

                                                          8

                                                          2+

                                                          5.32

                                                          9

                                                          10.9

                                                          PDAC

                                                          +

                                                          ND

                                                          -

                                                          +

                                                          -

                                                          No liver metastases on CT

                                                          9c,d

                                                          3+

                                                          2.95

                                                          6.2

                                                          35.0

                                                          PDAC

                                                          +

                                                          +

                                                          -

                                                          -

                                                          -

                                                          No liver metastases on CT

                                                          10e

                                                          3+

                                                          2.86*

                                                          6.3

                                                          27.0

                                                          PDAC

                                                          +

                                                          ND

                                                          -

                                                          -

                                                          -

                                                          No liver metastases on CT

                                                          Abbreviations: + present; - absent; COPD chronic obstructive pulmonary disease; CT computed tomography; FISH fluorescence in situ hybridization; IHC immunohistochemistry; ND not determined; PDAC pancreatic ductal adenocarcinoma.

                                                          aReceived adjuvant 5-fluouracil.

                                                          bReceived adjuvant gemcitabine.

                                                          cOnly case 9 received palliative chemotherapy (gemcitabine + erlotinib) and also received adjuvant gemcitabine.

                                                          dCase that was whole genome sequenced.

                                                          eCase 10 showed heterogeneous staining.

                                                          Table 3

                                                          Clinicopathological characteristics of HER2 amplified and HER2 non-amplified cases

                                                          Clinicopathological characteristics

                                                          Total cohort, n = 469

                                                          HER2 amplification

                                                          P-value

                                                            

                                                          Amplified, n = 10

                                                          Non-amplified, n = 459

                                                           

                                                          Sex, n (%)

                                                              

                                                           Male

                                                          241 (51)

                                                          6 (60)

                                                          220 (51)

                                                          0.7520b

                                                           Female 

                                                          228 (49)

                                                          4 (30)

                                                          210 (49)

                                                          Age, years

                                                              

                                                           Mean

                                                          66

                                                          64

                                                          66

                                                          0.5467c

                                                           Median

                                                          68

                                                          69.5

                                                          67.5

                                                           Range

                                                          28 to 88

                                                          47 to 73

                                                          28 to 87

                                                          AJCC stage, n (%)

                                                              

                                                           1a

                                                          17 (3.6)

                                                          1 (10)

                                                          16 (3.5)

                                                          0.4495d

                                                           1b

                                                          25 (5.3)

                                                          1 (10)

                                                          24 (5.2)

                                                           2a

                                                          132 (28.1)

                                                          3 (30)

                                                          129 (28)

                                                           2b

                                                          279 (59.5)

                                                          4 (40)

                                                          275 (60)

                                                           4

                                                          16 (3.4)

                                                          1 (10)

                                                          15 (3)

                                                          T stage, n

                                                              

                                                           T1

                                                          32

                                                          1

                                                          31

                                                           

                                                           T2

                                                          64

                                                          2

                                                          62

                                                           

                                                           T3

                                                          372

                                                          7

                                                          365

                                                           

                                                           T4

                                                          1

                                                          0

                                                          1

                                                           

                                                          N stage, n

                                                              

                                                           N0

                                                          180

                                                          6

                                                          174

                                                           

                                                           N1

                                                          289

                                                          4

                                                          185

                                                           

                                                          AJCC grade, n

                                                              

                                                           1 (well differentiated)

                                                          36

                                                          0

                                                          36

                                                          0.1259d

                                                           2 (moderately differentiated)

                                                          305

                                                          10

                                                          295

                                                           3 (poorly differentiated)

                                                          125

                                                          0

                                                          125

                                                           4 (undifferentiated)

                                                          3

                                                          0

                                                          3

                                                          Tumour size, mm

                                                              

                                                           <=20

                                                          105

                                                          3

                                                          102

                                                          0.4695b

                                                           >20

                                                          364

                                                          7

                                                          357

                                                          Vascular invasion, n

                                                              

                                                           Present

                                                          222

                                                          2

                                                          220

                                                          0.1106b

                                                           Absent

                                                          247

                                                          8

                                                          239

                                                          Perineural invasion, n

                                                              

                                                           Present

                                                          339

                                                          8

                                                          331

                                                          0.7336b

                                                           Absent

                                                          130

                                                          2

                                                          128

                                                          Tumour location, n

                                                              

                                                           Head

                                                          381

                                                          9

                                                          372

                                                          0.6961b

                                                           Others

                                                          88

                                                          1

                                                          87

                                                           Margins

                                                              

                                                           Clear

                                                          301

                                                          7

                                                          294

                                                          1.0000b

                                                           Involved

                                                          168

                                                          3

                                                          165

                                                          Outcome

                                                              

                                                           Follow-up, months 

                                                          0.03 to 240

                                                          5.0 to 43.6

                                                          0.03 to 240

                                                          1.0000b

                                                           Median follow-up, months 

                                                          16

                                                          23

                                                          16

                                                           Death: PDAC

                                                          369

                                                          8

                                                          361

                                                           Death: other

                                                          32

                                                          2

                                                          30

                                                           Death: unknown

                                                          16

                                                          0

                                                          16

                                                           Alive

                                                          49

                                                          0

                                                          49

                                                           Lost to follow-up

                                                          3

                                                          0

                                                          3

                                                          Cancer-specific survival, mean ± SD

                                                              

                                                           Length, months

                                                          20 ± 19.43

                                                          28 ± 19.65

                                                          20 ± 19.55

                                                          0. 2502c

                                                          Recurrence

                                                              

                                                           Present

                                                          261

                                                          8

                                                          253

                                                           

                                                           Absent

                                                          78

                                                          1

                                                          77

                                                           

                                                           Unknown

                                                          130

                                                          1

                                                          129

                                                           

                                                          Pattern of recurrence, n (% of total n)

                                                              

                                                           Lung without liver metastasis

                                                          22 (8.4% of 261)

                                                          4 (50% of 8)

                                                          18 (7.1% of 253)

                                                          0.0022b

                                                           Any recurrence without liver metastasis

                                                          127 (49% of 261)

                                                          8 (100% of 8)

                                                          119 (47% of 253)

                                                          0.0028b

                                                          Adjuvant therapy, n

                                                              

                                                           Yes

                                                          175

                                                          3

                                                          172

                                                           

                                                           No

                                                          289

                                                          7

                                                          282

                                                           

                                                           Unknown

                                                          5

                                                          0

                                                          5

                                                           

                                                          AJCC, American Joint Committee on Cancer; PDAC, pancreatic ductal adenocarcinoma.

                                                          aAJCC Cancer Staging Manual, seventh edition.

                                                          bFisher’s exact test.

                                                          cUnpaired t-test.

                                                          dχ2 test.

                                                          Since the completion of this study, we have begun prospectively screening patients with PDAC for HER2 amplification using this diagnostic approach, and identified a further two individuals (of eleven) with HER2 amplification. The first was a primary resected cancer with no evidence of recurrent disease to date at 6 months. Subsequently, whole genome sequence analysis on this patient’s tumour confirmed mutation in KRAS p.G12V . The second patient had an atypical pattern of metastatic disease at diagnosis, with a small primary pancreatic tumour on imaging, histologically confirmed peritoneal disease in the pelvis, and nodules in the lungs. Because of the unusual pattern of disease, PDAC was verified using a targeted exome panel of tissue from this patient’s peritoneal disease, which showed mutations in KRAS p.G12V and SMAD4, further supporting the diagnosis of PDAC.

                                                          HER2 amplification in other cancer types

                                                          The low prevalence of HER2 amplification in PDAC and many other cancer types makes clinical trials challenging. Analysis of genomic sequence data for 16 other cancer types (by TCGA, ICGC and other large-scale genomic efforts) identified an incidence of 0.5% to 13%, which equates to over 54,000 new cases of HER2-amplified cancers per year in the USA alone (see Additional file 10). Excluding breast and gastric cancer, we estimate an incidence in excess of 22,000 HER2 amplified cancers per annum. Recruiting patients based on HER2 amplification rather than organ of tumour origin would make trials of anti-HER2 therapies feasible in less common cancer types. In order to identify these 22,000 patients, 740,000 patients would need to be screened using IHC, and of these, all 2+ and 3+ IHC cases (~8%) would require validation using ISH. This approach may circumvent the challenges of previous clinical trials based on organ of origin recruitment, in which there have been mixed results but some efficacy in individuals (see Additional file 11). An orthogonal approach by which a primary molecular classification is used and patients are sub-classified based on organ of origin may be more tractable.

                                                          Discussion

                                                          In this study, we found that HER2-amplified PDAC has a prevalence of 2%, is detectable using contemporary genomic approaches, is associated with a clinical phenotype characterized by metastatic spread predominantly to the lungs and peritoneum with local recurrence, and can metastasize to the brain but tends to avoid the liver. It bears molecular similarities to HER2-amplified breast cancer, and is yet to be adequately assessed for potential responsiveness to anti-HER2 therapy. Multiple studies in large cohorts have shown that HER2-amplified breast cancers more commonly metastasize to the lung and the brain [22]. In PDAC, clinical trial data indicate that the first site of distant metastases occurred most commonly in the liver (50%), and occurred in the lung in 9% of the cases [23]. Autopsy studies have reported that 80% of distant metastases are to the liver, which occurred either alone or in combination with peritoneal and or lung metastases, while metastases sparing the liver made up the rest, and occurred in the peritoneum, lung, adrenal glands, and lymph nodes [24]. Cerebral metastases were not found in these studies. In our cohort of 469 patients, the incidence of lung metastases without liver metastases was 8%, comparable to previous studies. We detected only one case with brain metastases (0.2%), although only this single patient was investigated specifically for brain metastases. If we include all 10 HER2-amplified cases with documented metastatic disease, none had evidence of liver metastases (P = 0.0028), and the rate of lung metastases was 50% (P = 0.0022). These data suggest that HER2-amplified PDAC may have a distinct clinical phenotype, and that liver metastases are not determined by physical factors such as portal blood flow, but by the pathophysiology of disease.

                                                          These findings have significant clinical implications. First, the detection of small lung nodules should not delay the diagnosis of metastatic disease originating in the pancreas or at relapse if the liver and other sites are clear, particularly in known HER2-amplified cases. Second, if an individual is known to have an HER2-amplified PDAC, then monitoring for disease progression in non-traditional sites such as the lung and the brain, with vigilance for neurological symptoms may be prudent. Finally, there is potential for anti-HER2 therapies in this subset of patients.

                                                          In situ hybridization studies in our reference laboratory identified HER2 amplification only in PDACs with high protein expression by IHC (score 2+/3+). Therefore, a reasonable and cost-effective approach to universal HER2 screening is to initially test all cases with IHC and then perform secondary ISH testing only on cases with 2+/3+ staining, as was initially performed for breast cancer. Using this approach, 8% of PDAC (2+ and 3+ cancers) will require HER2 ISH assessment, and of these one-quarter will be amplified.

                                                          In the current study, in-depth genomic analysis, apart from HER2 amplification, did not reveal any features that are atypical of PDAC, with mutations of KRAS and TP53 and loss of SMAD4 found, although the inherent heterogeneity of PDAC makes it difficult to draw conclusions about the other mutations detected. mRNA expression profiles clustered with HER2-amplified breast cancer, suggesting that HER2 may be an important driver of carcinogenesis in this subgroup of PDAC.

                                                          It is interesting to note that all three HER2-amplified cases with available genomic data harboured the KRASp.G12V mutation. This mutation is less common than the p.G12D mutation, and accounts for 32% of KRAS mutations in PDAC versus 40% for p.G12D [9]. Given the small numbers of HER2-amplified cases, further studies of larger cohorts will be required before it can be determined if this association is sufficiently robust to be used diagnostically or targeted therapeutically.

                                                          Two clinical trials have assessed targeted trastuzumab therapy in PDAC [15, 16]. Both are single arm phase II trials used in combination with gemcitabine [15] and capecitabine [16]. Although the latter performed HER2 FISH for 2+ expressing cases, the former did not, and neither verified the 3+ IHC cases by FISH. In addition, these were not standardized assays performed in reference laboratories, and resulted in a HER2 positive rate of over 10%. This likely overestimation underpowered the trials by over 80%, making a negative result uninterpretable.

                                                          Conclusion

                                                          HER2 amplification occurs in 2.1% of PDAC cases, and is associated with an atypical pattern of metastatic disease. A number of cancers of different organs including NSCLC, ovarian cancer, cholangiocarcinoma, and PDAC have well-defined low-prevalence HER2 amplification. Testing anti-HER2 therapies may not be feasible in organ groups because of this low prevalence and the likely heterogeneous response rates. However, these studies could be approached using novel adaptive clinical trials testing personalized therapeutic strategies (for example,: BATTLE [25], I-SPY [26], FOCUS 4 [27] and IMPaCT [3]), or using a molecular taxonomy or ‘biotype’ that recruits HER2-amplified cancers irrespective of the organ in which the tumour arises (often referred to as ‘basket’ trials) [28], with specific attention to diagnostic criteria for patient recruitment, particularly as more effective anti-HER2 therapies emerge.

                                                          Abbreviations

                                                          APGI: 

                                                          Australian Pancreatic Genome Initiative

                                                          FISH: 

                                                          Fluorescence in situ hybridization

                                                          ICGC: 

                                                          International Cancer Genome Consortium

                                                          IHC: 

                                                          Immunohistochemistry

                                                          ISH: 

                                                          In situ hybridization

                                                          NSCLC: 

                                                          Non-small cell lung cancer

                                                          PDAC: 

                                                          Pancreatic ductal adenocarcinoma

                                                          SNP: 

                                                          Single nucleotide polymorphism

                                                          TCGA: 

                                                          The Cancer Genome Atlas.

                                                          Declarations

                                                          Funding/support

                                                          The work was supported by the National Health and Medical Research Council of Australia (NHMRC; 631701, 535903, 535914); Cancer Council NSW (SRP06-01; ICGC09-01; SRP11-01); Australian Government: Department of Innovation, Industry, Science, Research and Tertiary Education (DIISRTE); Australian Cancer Research Foundation (ACRF); Queensland Government (NIRAP); University of Queensland; Cancer Institute NSW (06/ECF/1-24, 09/CDF/2-40, 07/CDF/1-03, 10/CRF/1-01, 08/RSA/1-15, 10/CDF/2-26,10/FRL/2-03, 06/RSA/1-05, 09/RIG/1-02, 10/TPG/1-04, 11/REG/1-10, 11/CDF/3-26); Garvan Institute of Medical Research; Avner Nahmani Pancreatic Cancer Foundation; R.T. Hall Trust; Jane Hemstritch in memory of Philip Hemstritch; Gastroenterological Society of Australia (GESA); Royal Australasian College of Surgeons (RACS); Sydney Catalyst Translational Cancer Centre; Royal Australasian College of Physicians (RACP); Royal College of Pathologists of Australasia (RCPA) and the St Vincent’s Clinic Foundation.

                                                          Acknowledgments

                                                          We thank all the members of the Australian APGI (http://​www.​pancreaticcancer​.​net.​au/​apgi/​collaborators) for their continuing support with provision of biospecimens and clinical data. We also thank Mary-Anne Brancato BSc, Michelle Thomas BSc, Sarah Rowe BSc, and Mona Martyn-Smith BSc for maintenance of the APGI database and biospecimen resource.

                                                          Authors’ Affiliations

                                                          (1)
                                                          Kinghorn Cancer Centre and Garvan Institute of Medical Research
                                                          (2)
                                                          Anatomical Pathology, Sydpath, St Vincent’s Hospital
                                                          (3)
                                                          St Vincent’s Clinical School, University of New South Wales
                                                          (4)
                                                          Department of Anatomical Pathology, Royal North Shore Hospital, St Lenoards
                                                          (5)
                                                          Sydney Medical School, University of Sydney
                                                          (6)
                                                          Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland
                                                          (7)
                                                          Department of Cancer Medicine, Hammersmith Hospital, Imperial College Healthcare NHS Trust
                                                          (8)
                                                          Prince of Wales Clinical School, University of New South Wales and Prince of Wales Hospital
                                                          (9)
                                                          Histopath Pathology
                                                          (10)
                                                          Macarthur Cancer Therapy Centre, Sydney South West District Health Service
                                                          (11)
                                                          Upper Gastrointestinal Surgery Unit, Royal North Shore Hospital
                                                          (12)
                                                          Wolfson Wohl Cancer Research Centre, Institute of Cancer Sciences, University of Glasgow
                                                          (13)
                                                          West of Scotland Pancreatic Unit, Glasgow Royal Infirmary

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                                                          Copyright

                                                          © Chou et al.; licensee BioMed Central Ltd. 2013

                                                          This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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