In this first histopathological review of Morocco, we have investigated the prognostic value of CD44 cell-surface glycoprotein in 32 peripheral neuroblastic tumors and its relationship to other known prognostic indicators such as age at diagnosis, INSS stage of the disease, primary site, metastasis, differentiation and modified Shimada system.
An earlier study by Combaret et al. showed that CD44 expression strongly correlated with patient’s age as it was expressed on 61 of 66 tumors from infants (less than 1 year of age), but only in 47 of 74 tumors from older children (P < 0.0001)
. We were unable to demonstrate such a correlation probably because of the high mean age of our study cohort (41 months) while the frequent median age of pNTs diagnosis is 22 months
. This disparity may be due to a delay in diagnosis caused by difficulties in access to health care. Moreover, the study of Combaret et al., achieved in 1997, had investigated the CD44 expression compared to the 1 year cut-off, while a recent study showed evidence for an age cut-off (between 15 and 19 months) greater than 1 year for use in risk stratification of neuroblastoma patients
. So we definitely retained the threshold of 18 months in our study.
Study achieved by Taran et al. did not find a clear statistically significant correlation between CD44 expression and histoclinical parameters and currently known prognostic factors; however, this study had reported CD44 expression in 88.88% of cases and, unlike our study, it has noticed that the strongest CD44 expression was observed in tumors situated in the retroperitoneal space
As reported by several other studies
[31, 32], we found that CD44 is down-regulated in advanced neuroblastomas (stage III and IV), whereas the earlier and prognostically favorable stages (I, II, IVs) are characterized by tumor cells maintaining their ability to synthesize the standard form of CD44 (P = 0.006). This significant correlation of CD44 expression with the INSS stage strongly suggests that it might help in the prediction of clinical outcome. In contrast, another study demonstrated no major variation of CD44 incidence throughout the stages, except for stage IVs (I-II-III: 84%, IVs: l00%, IV: 85%)
. We can then speculate that CD44 might positively contribute to maturation and spontaneous regression of low grade stages I, II and IVs neuroblastic tumors.
In our study, absence of CD44s expression correlated significantly with a lack of differentiation (P = 0,0025). This result was comparable with other studies on neuroblastic tumors, indicating that the expression of CD44 standard form is linked to tumor maturation and differentiation
[34, 35]. These findings were supported by a study which reported that cultured human neuroblastoma GOTO cells, of which a cell adhesion molecule CD44 expression is usually suppressed, could be induced to differentiate into Schwannian cells and neuronal cells in the presence of 5-bromo-2’-deoxyuridine (BrdU) and by serum depletion respectively. These GOTO cells differentiated into Schwannian cells, specifically expressed CD44 glycoprotein, while this molecule remained suppressed in cells differentiated into neuronal cells which suggests that CD44 might play an important role in GOTO cells differentiation into Schwannian cells
 and thus in tumor evolution into maturing subtypes.
Our data analyzing the expression of CD44s in 32 neuroblastic tumours according to the INPC system (modified Shimada grading), demonstrated that 92.3% of CD44s-positive tumors were of a favorable prognostic type based on the INPC (with a better event-free survival probability) versus 68.4% CD44s-negative tumors in the unfavorable prognostic category (poorer event-free survival probability) (P = 0,001). These data were in agreement with another study in which approximately 89.5% of CD44s- negative pNTs were associated with the unfavorable prognostic INPC system in comparison with CD44s-positive tumors (approximately 65.5%) (P <0.05)
. Several other groups have shown the same correlation
[32, 35]. Therefore, absence of expression of CD44s can be seen in the more immature peripheral neuroblastic tumors, which correspond with the worst prognostic type based on the modified Shimada system
Cell adhesion is the condition sine qua non for the development of multicellular organisms. Cell migration, under physiological conditions, is the most important during embryogenesis, in tissue remodelling, wound healing and leukocyte migration
. Cell adhesion and migration are critical steps in cancer progression.
Simplistically, metastasis is depicted as the sequential dissociation of tumor cells from the primary tumor, migration of the dissociated cells into and through normal tissue, intravasation, survival in circulation, extravasation, migration into and through extracellular matrices, tumor cell proliferation, or, for tumor cells with high invasive potential, proliferation, invasion, and uncontrolled progression at this and/or multiple other sites
[38, 39]. In neuroblastic tumors, metastases are found mainly in the bone marrow, bone and lymph nodes
, however, other less frequent secondary sites such as the liver, kidneys, lungs and even the heart have been reported
. Many cell adhesion molecules including integrins, cadherins, immunoglobulin (Ig)-like CAMs, selectins, miscellaneous others and CD44, have been reported to be involved in each of these steps of the metastatic process and to be expressed by primary neuroblastoma cells or cell lines and associated with a given function or phenotype in this cell type
The role of CD44 adhesion glycoprotein in the evolution and progression of cancer has received a lot of attention in recent years
[38, 41]. In view of the fact that hyaluronic acid is the major component of the extracellular matrix and CD44 is the major receptor for HA
, it is not surprising that CD44 plays a main role in HA adhesion and crawling along the HA matrix thereby providing a mechanism by which CD44 could influence adhesion and de-adhesion to the extracellular matrix
. It is believed that elevated HA levels form a less dense matrix and consequently enhance cell motility as well as invasive ability into other tissues
. The HA–CD44 interactions have a central role in receptor tyrosine kinase (RTK)-induced activation of anti-apoptotic pathways and actively promote tumor cells and possibly cancer-initiating cell survival through their associations with multidrug resistance genes
. Importantly, activation of signalling pathways initiated by the tumor matrix could be inhibited by HA degradation, by competition with small HA fragments, by CD44 blockade or by CD44 knockdown
In most cancers, the dysregulated expression of CD44 is not the result of CD44 mutations. Instead, genes that are implicated in promoting carcinogenesis control the patterns of CD44 expression in cancer cells. Alternative splicing, for example, is under the control of mitogenic signals including the Ras-MAP kinase cascade. In addition, the loss of different subunits of the SWI/SNF chromatin remodeling complex, which are mutated in numerous cancers, results in the loss of CD44 transcription
. Aberrant CD44 expression is therefore inextricably linked to genetic alterations that lead to tumor growth and metastasis.
High expression of CD44 was observed on the surface of skin, cervix, endometrium, stomach, colon and prostate cancer cells
[22, 30]. It has been described in the literature that the presence of the CD44 molecule allows neoplastic cells to metastasize. However, results from other research have proved that it is not an increase but a decrease of CD44 expression that appears as an unfavorable prognostic factor in bladder cancer
Our data reported a statistical significant association (P = 0.013), between CD44 positive expression and the absence of metastasis, on human neuroblastic cells. A study by Valentiner et al. reported that expression of CD44 was associated with a metastatic pattern of the neuroblastoma cell lines engrafted in the SCID mice and that CD44-negative neuroblastomas developed numerous micrometastases in the lung interstitium while CD44-positive neuroblastomas produced multicellular metastases predominantly located in the intra- and periarterial space of the lung
. These data have been contradicted by a more recent report showing that the CD44 negative SK-N-SH neuroblastoma cells were the ones that infiltrated the bone marrow, spleen and liver of transplanted animals, clearly indicating that the metastatic ability of neuroblastoma cells is independent of CD44
. This study also showed that lack of CD44 expression was accompanied by lower levels of various adhesion molecules, including CD49d (α4 integrin), CD49e (α5 integrin) and CD29 (β1 integrin), as well as ICAM1 and neural cell adhesion molecule NCAM
 which reduce adherence capacity and may enhance the cells migratory ability and their propensity to form metastases
. Therefore, the distinct cell adhesion profile of CD44s negative cells suggests an enhanced metastatic potential.