Mohammed A. Aleskandarany

The prognostic significance of inflammation and medullary histological type in invasive carcinoma of the breast

UNLABELLED The new gene expression molecular taxonomy of breast cancer places medullary carcinoma in the basal group. The basal group is considered to have a poor prognosis, but medullary carcinoma is considered to have a better prognosis than other grade 3 carcinomas. The prognostic significance of tumour associated inflammation, an important feature of medullary carcinomas, remains controversial. The aim of this study was to assess the prognostic importance of medullary histological type and inflammation in breast cancer. One thousand five hundred and ninety-seven patients who received no systemic adjuvant treatment and who had a median follow up of 9.5 years were studied. RESULTS Prominent inflammation was associated with high histological grade and with better survival [relative risk (RR) 0.57, 95% confidence intervals (CI) 0.44-0.74] on multivariate analysis. Typical and atypical medullary carcinomas (n=132) did not have significantly different survival and were grouped together. Medullary carcinoma did not have significantly different prognosis than grade 3 ductal carcinoma with prominent inflammation, but both had a better prognosis than grade 3 ductal carcinoma without prominent inflammation (P<0.0001 and P=0.03). These differences were independent of other prognostic factors. These results question the current separation of typical and atypical medullary carcinoma. Prominent inflammation is associated with a better prognosis, and may explain the better prognosis in medullary carcinoma compared with grade 3 ductal carcinoma without prominent inflammation. The good prognosis of medullary carcinoma emphasises the heterogeneity of basal-like breast carcinomas. Further studies are needed to investigate the difference in survival between medullary carcinoma and other forms of basal carcinomas and the role of inflammation in any such differences in behaviour.

Prognostic value of proliferation assay in the luminal, HER2-positive, and triple-negative biologic classes of breast cancer

IntroductionAlthough the prognostic significance of proliferation in early invasive breast cancer has been recognized for a long time, recent gene-expression profiling studies have reemphasized its biologic and prognostic value and the potential application of its assessment in routine practice, particularly to define prognostic subgroups of luminal/hormone receptor-positive (HR+) tumors. This study aimed to assess the prognostic value of a proliferation assay by using Ki-67 immunohistochemistry as compared with mitotic count scores.MethodProliferation was assessed by using Ki-67 labeling index (Ki-67LI) and mitotic scores in a large (n = 1,550) and well-characterized series of clinically annotated primary operable invasive breast cancer with long-term follow-up. Tumors were phenotyped based on their IHC profiles into luminal/HR+, HER2+, and triple-negative (TN) classes. We used a split-sample development and validation approach to determine the optimal Ki-67LI cut-offs.ResultsThe optimal cut-points of Ki-67LI were 10% and 50% for the luminal class. Both Ki7LI and MS were able to split luminal tumors into subgroups with significantly variable outcomes, independent of other variables. Neither mitotic count scores nor Ki-67LI was associated with outcome in the HER2+ or the TN classes.ConclusionsAssessment of proliferation by using Ki-67LI and MS can distinguish subgroups of patients within luminal/hormone receptor-positive breast cancer significantly different in clinical outcomes. Overall, both Ki-67 LI and mitotic-count scores showed comparable results. The method described could provide a cost-effective method for prognostic subclassification of luminal/hormone receptor-positive breast cancer in routine clinical practice.

Growth fraction as a predictor of response to chemotherapy in node-negative breast cancer

Accurate predictive markers of chemotherapeutic response in early breast cancer are still lacking. The role of tumour growth fraction as a predictor of response to chemotherapy was assessed in early breast cancer. In this study, immunohistochemical expression of MIB1 was studied in a well‐characterised series of early (Stages I and II) node‐negative breast carcinoma cases (n = 100) with long‐term follow‐up that have received adjuvant chemotherapy (cyclophosphamide/methotrexate/5‐fluorouracil regimen). In addition, 728 cases who did not receive adjuvant chemotherapy were used as a control group. Increased tumour growth fraction was associated with a better response to adjuvant chemotherapy in terms of longer breast cancer specific survival and disease‐free interval [hazard ratio (HR) = 0.354, 95% CI = 0.177–0.688, p = 0.003 and HR = 0.396, 95% CI = 0.205–0.768, p = 0.006, respectively]. In contrast to the control group, patients with high growth fraction tumour (>70%) showed an excellent outcome with infrequently reported events during the period of follow‐up. Importantly, patients with a low growth fraction (≤10%) showed frequent recurrences and shorter survival time with outcome comparable to those of high growth fraction who did not receive chemotherapy. Therefore, tumour growth fraction can be used to assign patients into distinct groups showing differential response to adjuvant chemotherapy. Patients with a high growth fraction appear to be ideal candidates for adjuvant chemotherapy while those with low growth fraction are less likely to benefit and are prone to the potential serious side effects of adjuvant chemotherapy.

Cten signals through integrin-linked kinase (ILK) and may promote metastasis in colorectal cancer

CTEN/TNS4 is an oncogene in colorectal cancer (CRC), which can induce cell motility although its mechanistic basis of activity and the clinical implications of Cten expression are unknown. As Cten is in complex with integrins at focal adhesions, we hypothesised that it may interact with integrin-linked kinase (ILK). Through forced expression and knockdown of Cten in HCT116 and SW620 (respectively, showing low and high Cten expression), we showed that Cten could regulate ILK. However, inhibition of ILK after forced expression of Cten abrogated the motility-inducing effects of Cten, thereby demonstrating that the Cten–ILK interaction was functionally relevant. Combined knockdown of Cten and ILK had no additive effects on cell motility compared with knockdown of each individually. In order to investigate the clinical implications of Cten expression, a series of 462 CRCs were evaluated by immunohistochemistry. High expression of Cten was associated with advanced Dukes’ stage (P<0.001), poor prognosis (P<0.001) and distant metastasis (P=0.008). The role of Cten in metastasis was tested by (a) intrasplenic injection of CRC cells stably transfected with a Cten expression vector into nude mice and (b) testing a series of primary human CRCs and their metastases by immunohistochemistry. Compared with controls, mice injected with cells expressing Cten developed larger tumours in the spleen (P<0.05) and liver (P<0.05). In the human cases, compared with primary tumours, the metastatic deposits had a significantly higher frequency of nuclear localisation of Cten (P=0.002). We conclude that Cten expression is of prognostic significance in CRC, and we delineate a Cten–ILK pathway controlling cell motility and possibly promoting metastasis.

An approach to the diagnosis of spindle cell lesions of the breast

Although most breast spindle cell lesions (BSCLs) are rare, they constitute a wide spectrum of diseases, ranging from reactive processes to aggressive malignant tumours. Despite their varied histogenesis and behaviour, some lesions show an overlap of morphological features, making accurate diagnosis a challenging task, particularly in needle core biopsies. Clinical history and immunohistochemistry can help in making a correct diagnosis in morphologically challenging cases. To make an accurate diagnosis, it is important to maintain a wide differential diagnosis and be familiar with the diverse morphological appearances of these different entities. BSCLs can generally be classified into bland‐looking and malignant‐looking categories. In the former, the commonest diagnosis is scarring. However, it is important to distinguish low‐grade spindle cell metaplastic breast carcinoma from other benign entities, as the management is clearly different. In the malignant category, it is important to differentiate metaplastic carcinoma from other malignant primary and metastatic malignant spindle cell tumours of the breast, such as malignant phyllodes tumour, angiosarcoma, and melanoma. This review focuses on the classification and histological and molecular diagnosis of various BSCLs, with an emphasis on the diagnostic approach, including in core biopsies.

MYC functions are specific in biological subtypes of breast cancer and confers resistance to endocrine therapy in luminal tumours

Background:MYC is amplified in approximately 15% of breast cancers (BCs) and is associated with poor outcome. c-MYC protein is multi-faceted and participates in many aspects of cellular function and is linked with therapeutic response in BCs. We hypothesised that the functional role of c-MYC differs between molecular subtypes of BCs.Methods:We therefore investigated the correlation between c-MYC protein expression and other proteins involved in different cellular functions together with clinicopathological parameters, patients’ outcome and treatments in a large early-stage molecularly characterised series of primary invasive BCs (n=1106) using immunuohistochemistry. The METABRIC BC cohort (n=1980) was evaluated for MYC mRNA expression and a systems biology approach utilised to identify genes associated with MYC in the different BC molecular subtypes.Results:High MYC and c-MYC expression was significantly associated with poor prognostic factors, including grade and basal-like BCs. In luminal A tumours, c-MYC was associated with ATM (P=0.005), Cyclin B1 (P=0.002), PIK3CA (P=0.009) and Ki67 (P<0.001). In contrast, in basal-like tumours, c-MYC showed positive association with Cyclin E (P=0.003) and p16 (P=0.042) expression only. c-MYC was an independent predictor of a shorter distant metastases-free survival in luminal A LN+ tumours treated with endocrine therapy (ET; P=0.013). In luminal tumours treated with ET, MYC mRNA expression was associated with BC-specific survival (P=0.001). In ER-positive tumours, MYC was associated with expression of translational genes while in ER-negative tumours it was associated with upregulation of glucose metabolism genes.Conclusions:c-MYC function is associated with specific molecular subtypes of BCs and its overexpression confers resistance to ET. The diverse mechanisms of c-MYC function in the different molecular classes of BCs warrants further investigation particularly as potential therapeutic targets.

Camptothecin targets WRN protein: mechanism and relevance in clinical breast cancer

Werner syndrome protein (WRN) is a RecQ helicase that participates in DNA repair, genome stability and cellular senescence. The five human RecQ helicases, RECQL1, Bloom, WRN, RECQL4 and RECQL5 play critical roles in DNA repair and cell survival after treatment with the anticancer drug camptothecin (CPT). CPT derivatives are widely used in cancer chemotherapy to inhibit topoisomerase I and generate DNA double-strand breaks during replication. Here we studied the effects of CPT on the stability and expression dynamics of human RecQ helicases. In the cells treated with CPT, we observed distinct effects on WRN compared to other human RecQ helicases. CPT altered the cellular localization of WRN and induced its degradation by a ubiquitin-mediated proteasome pathway. WRN knockdown cells as well as CPT treated cells became senescent and stained positive for senescence-associated β-galactosidase at a higher frequency compared to control cells. However, the senescent phenotype was attenuated by ectopic expression of WRN suggesting functional implication of WRN degradation in CPT treated cells. Approximately 5-23% of breast cancer tumors are known to respond to CPT-based chemotherapy. Interestingly, we found that the extent of CPT-induced WRN degradation correlates with increasing sensitivity of breast cancer cells to CPT. The abundance of WRN decreased in CPT-treated sensitive cells; however, WRN remained relatively stable in CPT-resistant breast cancer cells. In a large clinical cohort of breast cancer patients, we find that WRN and topoisomerase I expression correlate with an aggressive tumor phenotype and poor prognosis. Our novel observations suggest that WRN abundance along with CPT-induced degradation could be a promising strategy for personalizing CPT-based cancer chemotherapeutic regimens.

Human Helicase RECQL4 Drives Cisplatin Resistance in Gastric Cancer by Activating an AKT–YB1–MDR1 Signaling Pathway

Elevation of the DNA-unwinding helicase RECQL4, which participates in various DNA repair pathways, has been suggested to contribute to the pathogenicity of various human cancers, including gastric cancer. In this study, we addressed the prognostic and chemotherapeutic significance of RECQL4 in human gastric cancer, which has yet to be determined. We observed significant increases in RECQL4 mRNA or protein in >70% of three independent sets of human gastric cancer specimens examined, relative to normal gastric tissues. Strikingly, high RECQL4 expression in primary tumors correlated well with poor survival and gastric cancer lines with high RECQL4 expression displayed increased resistance to cisplatin treatment. Mechanistic investigations revealed a novel role for RECQL4 in transcriptional regulation of the multidrug resistance gene MDR1, through a physical interaction with the transcription factor YB1. Notably, ectopic expression of RECQL4 in cisplatin-sensitive gastric cancer cells with low endogenous RECQL4 was sufficient to render them resistant to cisplatin, in a manner associated with YB1 elevation and MDR1 activation. Conversely, RECQL4 silencing in cisplatin-resistant gastric cancer cells with high endogenous RECQL4 suppressed YB1 phosphorylation, reduced MDR1 expression, and resensitized cells to cisplatin. In establishing RECQL4 as a critical mediator of cisplatin resistance in gastric cancer cells, our findings provide a therapeutic rationale to target RECQL4 or the downstream AKT-YB1-MDR1 axis to improve gastric cancer treatment. Cancer Res; 76(10); 3057-66. ©2016 AACR.

Chk1 phosphorylated at serine345 is a predictor of early local recurrence and radio-resistance in breast cancer

Radiation‐induced DNA damage activates the DNA damage response (DDR). DDR up‐regulation may predict radio‐resistance and increase the risk of early local recurrence despite radiotherapy in early stage breast cancers. In 1755 early stage breast cancers, DDR signalling [ATM, ATR, total Ckh1, Chk1 phosphorylated at serine345 (pChk1), Chk2, p53], base excision repair [PARP1, POLβ, XRCC1, FEN1, SMUG1], non‐homologous end joining (Ku70/Ku80, DNA‐PKcs) and homologous recombination [RAD51, BRCA1, γH2AX, BLM, WRN, RECQL5, PTEN] protein expression was correlated to time to early local recurrence. Pre‐clinically, radio‐sensitization by inhibition of Chk1 activation by ATR inhibitor (VE‐821) and inhibition of Chk1 (V158411) were investigated in MDA‐MB‐231 (p53 mutant) and MCF‐7 (p53 wild‐type) breast cancer cells. In the whole cohort, 208/1755 patients (11.9%) developed local recurrence of which 126 (61%) developed local recurrence within 5 years of initiation of primary therapy. Of the 20 markers tested, only pChk1 and p53 significantly associated with early local recurrence (p value = 0.015 and 0.010, respectively). When analysed together, high cytoplasmic pChk1‐nuclear pChk1 (p = 0.039), high cytoplasmic pChk1‐p53 (p = 0.004) and high nuclear pChk1‐p53 (p = 0.029) co‐expression remain significantly linked to early local recurrence. In multivariate analysis, cytoplasmic pChk1 level independently predicted early local recurrence (p = 0.025). In patients who received adjuvant local radiotherapy (n = 949), p53 (p = 0.014) and high cytoplasmic pChk1‐p53 (p = 0.017) remain associated with early local recurrence. Pre‐clinically, radio‐sensitisation by VE‐821 or V158411 was observed in both MCF‐7 and MDA‐MB‐231 cells and was more pronounced in MCF‐7 cells. We conclude that pChk1 is a predictive biomarker of radiotherapy resistance and early local recurrence.

Transcriptomic and Protein Expression Analysis Reveals Clinicopathological Significance of Bloom Syndrome Helicase (BLM) in Breast Cancer

Bloom syndrome helicase (BLM) has key roles in homologous recombination repair, telomere maintenance, and DNA replication. Germ-line mutations in the BLM gene causes Bloom syndrome, a rare disorder characterized by premature aging and predisposition to multiple cancers, including breast cancer. The clinicopathologic significance of BLM in sporadic breast cancers is unknown. We investigated BLM mRNA expression in the Molecular Taxonomy of Breast Cancer International Consortium cohort (n = 1,950) and validated in an external dataset of 2,413 tumors. BLM protein level was evaluated in the Nottingham Tenovus series comprising 1,650 breast tumors. BLM mRNA overexpression was significantly associated with high histologic grade, larger tumor size, estrogen receptor–negative (ER−), progesterone receptor–negative (PR−), and triple-negative phenotypes (ps < 0.0001). BLM mRNA overexpression was also linked to aggressive molecular phenotypes, including PAM50.Her2 (P < 0.0001), PAM50.Basal (P < 0.0001), and PAM50.LumB (P < 0.0001) and Genufu subtype (ER+/Her2−/high proliferation; P < 0.0001). PAM50.LumA tumors and Genufu subtype (ER+/Her2−/low proliferation) were more likely to express low levels of BLM mRNA (ps < 0.0001). Integrative molecular clusters (intClust) intClust.1 (P < 0.0001), intClust.5 (P < 0.0001), intClust.9 (P < 0.0001), and intClust.10 (P < 0.0001) were also more likely in tumors with high BLM mRNA expression. BLM mRNA overexpression was associated with poor breast cancer–specific survival (BCSS; ps < 0.000001). At the protein level, altered subcellular localization with high cytoplasmic BLM and low nuclear BLM was linked to aggressive phenotypes. In multivariate analysis, BLM mRNA and BLM protein levels independently influenced BCSS. This is the first and the largest study to provide evidence that BLM is a promising biomarker in breast cancer. Mol Cancer Ther; 14(4); 1057–65. ©2015 AACR.