Morphological and molecular characteristics of HER2 amplified urothelial bladder cancer

Specific genetic alterations in cancers may be associated with particular morphological features as shown in colon and prostate cancer [20, 21]. In UBC, there is currently only one comparable investigation. Ching et al. [22] evaluated the particularly aggressive micropapillary variant of urothelial carcinoma for HER2 aberrations [22] and demonstrated HER2 protein overexpression (score 2+ or 3+) in 68 % and HER2 gene amplification in 42 % of their 20 tumors. However, the micropapillary variant with a prevalence of 0.6–6 % is rare in UBC [17, 22, 23], and in general, a component of a conventional UBC. As yet, the morphological spectrum of UBC with HER2 amplification, which is an aggressive and from a therapeutic point of view a potentially distinct subgroup, has not been described. Better knowledge about its morphology might help to better identify these tumors. Therefore, we first identified HER2 amplified UBC in our cohort of high-risk, metastasizing tumors and subsequently described their morphology and HER2 expression patterns.

Micropapillary tumor growth was present in 77 % of our HER2 amplified tumors; 31 % were purely or predominantly micropapillary UBC, the residual tumors had minor micropapillary fractions. The proportion of micropapillary growth in relation to the entire tumor mass in this group was 33 %. This contrasts significantly with the HER2 non-amplified UBC group, in which micropapillary growth was present in only one tumor and this component occupied just 3 % of the entire evaluated tumor mass in this group. In addition, the HER2 amplified tumors presented with a significantly higher morphological heterogeneity than the control group, reflected by a higher number of subtype components per tumor, and showed a significantly higher tumor-associated chronic inflammatory infiltrate. Interestingly, the latter is in line with a recent study showing that HER2 overexpression activates multiple inflammatory pathways, especially NF-κB, which is critical to Interleukin-6 (Il-6) expression [24]. Whether or not a relationship exists with the more recently described association of polyoma virus with micropapillary UBC needs further investigation [25, 26]. Taken together, these histomorphological features of HER2 amplified UBC—frequent micropapillary architecture, morphological heterogeneity and marked tumor-associated chronic inflammation—allow pathologists to better identify this clinically important, particularly aggressive subset of UBC [12] and herewith to contribute to enhanced survival prediction and preselection for potential anti-HER2 therapies in the future.

Only few studies have investigated HER2 amplification and overexpression simultaneously in all their bladder cancer patients [27‐29]. However, they only categorized the tumors according to DAKO scores and did not report the exact percentage and distribution of positive tumor cells. We noted a strong complete membranous immunoreactivity for HER2 in 77 % of our HER2 amplified tumors. Importantly, most of these tumors showed this staining in virtually all neoplastic cells; only two cases had a partial tumor staining of 60 and 50 % of the tumor cells. This suggests that HER2 overexpression mostly occurs as an early event in tumorigenesis and only rarely in subsequent tumor development. Further evidence for early HER2 overexpression in tumorigenesis was noted in the two patients with residual urothelial carcinoma in situ which were strongly HER2 positive (score 3+). Interestingly, even though our HER2 amplified group presented with marked heterogeneous morphology, this observation was not reflected in the HER2 expression pattern. There was no HER2 expression difference between the morphologically diverse components of these tumors; in particular, the micropapillary areas did not show a more pronounced immunoreactivity than the other histological components. Notably, 23 % of our HER2 amplified tumors were immunohistochemically HER2 negative. Similarly, high rates of “false negative” UBC (20–24 %) have been reported by others [30, 29] and were attributed to putative fixation artifacts. Therefore, we specifically investigated HER2 expression intensity along the gradient of diffusion of formalin from the superficial bladder wall to deeper parts. However, there were no increments in staining intensities to be found.

Finally, we tested all UBC for activating HER2 mutations which have been reported in a small subset of lung and breast cancer [19, 31, 32]. Interestingly, we detected a D769N mutation in a HER2 amplified tumor sample. To our knowledge, this mutation has not been described so far, also not in the series of Ross et al who tested 15 micropapillary UBC for mutations [33]. However, two mutations, D769H and D769Y, occurring at the same amino acid position were described in breast cancer [31]. Both mutations conferred constitutive activity of the HER2 kinase. Cell lines bearing these mutations revealed increased HER2, EGFR, and PLCγ phosphorylation and had more rapid tumor growth in xenograft models compared to the wild type control. In addition, both mutations conferred sensitivity to the HER2 inhibitor lapatinib [31]. The authors suggested that activation of HER2 by these mutations may be due to loss of the acidic side chain at D769, or alternatively, due to an aromatic ring introduced by histidine or tyrosine, respectively. Asparagine and tyrosine can often been substituted without affecting protein function since both amino acids contain uncharged polar side chains. If D769N mutation, like D769Y mutation, induces HER2 activity, has to be confirmed experimentally.