Purpose According to the WHO, the cribriform pattern is a subtype of acinar (Aci) predominance in invasive adenocarcinoma (ADC) of the lung. Recently, several studies have demonstrated poor prognosis in patients with cribriform predominance. This study was performed to examine the correlations of cribriform pattern with the clinicopathology, molecular features and prognosis in patients with invasive ADC.
Methods Histological subtypes were evaluated in 279 patients who underwent complete resection for invasive ADC. Patients of the Aci-predominant subtype were divided into two subgroups according to the percentage of cribriform cancer (≥5% vs <5%). Clinicopathological characteristics, overall survival (OS), disease-free survival (DFS) and molecular changes were compared. In addition, both OS and DFS were compared between patients with cribriform-predominant (n=33) and pure Aci-predominant (n=88) ADCs.
Results A cribriform pattern was found in 111 (39.8%) cases and ranged from 5 % to 100 % of the total tumour volume (mean±SEM, 30%±2%). Of 117 patients with Aci predominance, 79 showed the cribriform pattern, while the remaining 38 did not. The cribriform pattern was associated with aggressive pathological behaviour, including advanced stages of cancer, nuclear atypia, mitoses, lymph node invasion, metastasis and larger tumour size. The subgroup with cribriform cancer (≥5%) had significantly poorer OS and DFS compared with the cribriform-negative (<5%) group. In addition, Cox multivariate analyses revealed that the cribriform pattern was an independent predictor of OS but not DFS. Moreover, OS was significantly lower in the cribriform-predominant group than in the Aci-predominant group.
Conclusion The cribriform pattern is associated with aggressive pathological behaviour and is an independent poor prognostic indicator in patients with Aci-predominant ADC of the lung.
- lung adenocarcinoma
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Lung cancer is the most common type of cancer worldwide, with pulmonary adenocarcinoma (ADC) being the most common histological type.1 2 Morphologically, pulmonary ADC is heterogeneous and is defined as a mixed subtype.3 Invasive ADC can be divided into lepidic (Lep), acinar (Aci), papillary (Pap), solid (Sol) and micropapillary (Mip) standard subtypes, or invasive mucinous, colloid, foetal and enteric variants, as established by the International Association for the Study of Lung Cancer (IASLC), the American Thoracic Society (ATS) and the European Respiratory Society (ERS).4 Numerous studies have investigated the possible associations between these new subtype classifications and prognosis of lung ADC.5–7 Further, there is accumulating evidence for additional growth morphologies with distinct clinical prognoses.8–11 Of these, the most prominent is the cribriform pattern, defined as invasive back-to-back fused tumour glands with poorly formed glandular spaces lacking intervening stroma or invasive nests of tumour cells that produce glandular lumina without solid components.9 Notably, Warth et al 10 reported that cases of cribriform-predominant ADC had poorer outcomes than Aci-predominant or Pap-predominant ADC. In this study, we investigated the correlations between the cribriform pattern and the clinical pathological characteristics, molecular changes and survival in Aci-predominant ADC. We also examined whether the cribriform-predominant type can be distinguished from the Aci-predominant subtype.
Materials and methods
This retrospective study included cases of lung ADC consecutively resected between October 2012 and December 2018 at the Department of Thoracic Surgery, The Second Affiliated Hospital of Zhengzhou University. To focus on our main questions and eliminate possible confounding variables, patients were required to meet the following inclusion criteria: complete resection with curative intent, pathologically confirmed lung ADC with at least 50% tumour structures and sufficient tissue for subtype analyses, solitary pure invasive ADC, and complete clinical pathology information. The exclusion criteria were history of other malignant tumours and preoperative assisted chemoradiotherapy. Tumour, node, metastases(TNM) stage was assigned based on the definitions in the eighth edition of the American Joint Committee on Cancer TNM Staging Manual.12
All available H&E-stained tumour slides (mean, 5 slides per patient; range, 1–12 slides per patient) were reviewed by two pathologists who were blinded to the clinical data of the patients, using an Olympus 300–2 microscope (Olympus, Tokyo, Japan) with a standard 22 mm diameter eyepiece. The criteria for evaluation followed the IASLC/ATS/ERS classification of invasive ADC and 2015 WHO guidelines for tumours of the lung.3 4 Comprehensive evaluation of histological subtyping was performed in a semiquantitative manner by recording the percentage of each histological subtype in 5% increments, totalling 100% for each tumour, as described elsewhere.3 4 The morphological structure with the highest percentage was designated as the predominant pattern: Lep-predominant, Pap-predominant, Aci-predominant, Sol-predominant and Mip-predominant; all other patterns (eg, secondary or tertiary) were considered minor components. Moreover, we noted that the cribriform pattern showed pathological features distinct from the five standard types of ADC. In the present study, the cribriform pattern included classical cribriform and variant cribriform (glomeruloid pattern and beaded pattern). Based on the report of Kadota et al ,9 we modified the definition of classical cribriform pattern to invasive, back-to-back fused tumour glands with poorly formed glandular spaces lacking intervening stroma. We defined one variant cribriform pattern as a glomeruloid pattern in which tumour cells protruded into the Aci lumen, forming several microcribriform structures, but were not attached to the other side of the Aci wall. The other variant cribriform pattern was defined as a beaded pattern in which several fused glands were arranged in a row or a ring. Representative images are shown in figure 1. Cribriform-predominant lung ADC was defined as ADC composed mostly of the cribriform pattern or tumours in which the cribriform component accounted for the highest percentage, similar to the above criteria.
Nuclear features were examined using a high-power field (HPF) microscope (0.237 mm2 field of view). Nuclear atypia was identified in the area with the greatest degree of atypia and was classified as follows: mild (nuclei uniform in size and shape), moderate (nuclei intermediate in size with slight irregularities) and severe (nuclei enlarged to varying degrees with some nuclei at least twice as large as others). Mitoses were assayed in at least 50 HPFs in areas with the highest mitotic activity. The number of mitoses was calculated as the average number of mitotic figures per 10 HPFs. According to the mitotic count, tumours were graded as follows: low, 0–1 mitotic figures per 10 HPFs; intermediate, 2–4 mitotic figures per 10 HPFs; and high, ≥5 mitotic figures per 10 HPFs.13–15 The following clinicopathological characteristics were also investigated: tumour size, metastasis, lymphatic and vascular invasions, and tumour spread through air spaces (STAS).16
Correlations between two categorical variables were tested using Pearson’s χ2 test or Fisher’s exact test. Survival analyses were estimated using the Kaplan-Meier method, and nonparametric group comparisons were performed using the log-rank test. The Cox proportional hazards regression method was employed in multivariate analyses to identify independent prognostic factors. All p values were based on two-tailed statistical analyses, and p<0.05 was considered statistically significant. Statistical analyses were conducted using SPSS V.21 and GraphPad Prime V.7.0. DFS and OS were recorded during follow-up clinic visits or by telephone.
Important clinicopathological findings of all patients are summarised in online supplementary table 1. The cohort consisted of 279 patients with resected stages I–III invasive ADC of the lung. Median follow-up was 36 months (range 1.1–116.2 months) and the median age was 58 years (range, 27–83 years). There were slightly more men (n=147) than women (n=132) enrolled in the study. Ninety of the male patients had a history of smoking or were current smokers compared with only one of the women. Most patients (n=194) had a clinically advanced stage of cancer. During the study period, 77 patients experienced recurrence or disease progression and 141 died after surgery. Microscopic observation of the state of STAS (54.1%, n=151), lymphatic invasion (41.2%, n=115), vascular invasion (21.1%, n=59) and necrosis (25.1%, n=70) was conducted. With respect to the predominant growth pattern, all 279 cases were further divided into five predominant subtypes as per the 2015 WHO classification criteria3: Lep (22, 7.9%), Pap (45, 16.1%), Aci (including cribriform-predominant) (117, 41.9%), Sol (76, 27.3%) and Mip (19, 6.8%) (figure 2). In all tumours (n=279), the cribriform pattern (≥5%) was strongly associated with lymphatic invasion (p=0.001), nuclear atypia (p<0.001) and mitosis (p<0.001) (online supplementary table 2).
Associations between the cribriform pattern and clinicopathological characteristics
The cribriform pattern was present in 111 (39.8%) cases, ranging from 5% to 100% of the total tumour volume (mean±SEM, 30%±2%). The frequency of the cribriform pattern was highest in Aci-predominant ADC (79 of 117, 67.5%), followed by Sol-predominant (41 of 76, 53.9%), Pap-predominant (19 of 45, 42.2%), Mip-predominant (7 of 19, 36.8%) and Lep-predominant (1 of 22, 4.5%) ADCs (p<0.001). In addition, 33 cribriform-predominant cases (11.8%) had formerly been classified as Aci-predominant. As expected, the cribriform pattern was more frequently identified in patients with an advanced clinical stage of cancer (p=0.004), lymph node invasion (p=0.018), metastasis (p=0.004) and larger tumour sizes (p=0.020). Meanwhile, nuclear atypia and nuclear mitosis were more pronounced (p=0.013 and p<0.001, respectively) in tumours with cribriform pattern (table 1).
Association between the cribriform pattern and prognosis
We divided the tumours into two subgroups based on the percentage of the cribriform component (≥5% and<5%) in the Aci-predominant cohort (n=117). Patient survival was significantly lower (OS, p<0.001; DFS, p=0.005) in the ≥5% group (n=79) than in the <5% group (n=38) (table 2, figure 3A,B). Advanced clinical stage (OS, p=0.005; DFS, p<0.001), lymph node invasion (OS, p=0.006; DFS, p<0.001), tumour metastasis (OS, p<0.001; DFS, p<0.001), more pronounced nuclear atypia (OS, p=0.006; DFS, p=0.008), more mitoses (OS, p<0.001; DFS, p=0.006), presence of tumour necrosis (OS, p=0.036; DFS, p=0.020) and presence of STAS (OS, p=0.049; DFS, p<0.001) were negative prognostic factors in univariate analyses. However, vascular invasion was significantly associated with poorer DFS (p=0.017) but not OS (p=0.151). Multivariate analyses showed that cribriform pattern (≥5%) (HR 2.768, 95% CI 1.101 to 6.956, p=0.030), more mitotic figures (HR 1.692, 95% CI 1.112 to 2.575, p=0.014) and distant metastasis (HR 2.192, 95% CI 1.112 to 4.323, p=0.023) were independent predictors of OS. The STAS condition (HR 1.826, 95% CI 1.023 to 3.259, p=0.042) and tumour metastasis (HR 1.938; 95% CI 1.007 to 3.728, Pp=0.047) were independent factors of DFS (table 3).
When the cribriform pattern was considered as a separate pattern in histological analyses, patients with cribriform-predominant tumours (n=33) remained at higher risk with regard to OS compared with patients with pure Aci-predominant (n=84, p=0.035), Sol-predominant (n=76, p=0.268) and Mip-predominant (n=19, p=0.514) tumours (figure 3C). Meanwhile, the DFS of patients with the cribriform-predominant pattern was also similar to those of patients with Sol-predominant (n=79, p=0.921) or Mip-predominant (n=19, p=0.701) tumours (figure 3D).
To exclude the effects of cribriform-predominant tumours (n=33), we restricted survival analyses to patients with pure Aci-predominant tumours (n=84). In this cohort, the cases were further divided into two groups based on cribriform percentage (≥5% and <5%). Patients in the ≥5% group showed unfavourable clinical outcomes compared with their counterparts (OS, p=0.001; DFS, p=0.006) (figure 3E,F). The risk indicators of the primary tumour were related to advanced stage of cancer (OS, p=0.017; DFS, p<0.001), lymph node metastasis (OS, p=0.033; DFS, p<0.001), tumour metastasis (OS, p<0.001; DFS, p=0.001) and frequent mitosis (OS, p<0.001; DFS, p=0.007) (online supplementary table 1). Multivariate survival analyses incorporating the above variables showed that advanced mitosis (HR 2.189, 95% CI 1.327 to 3.609, p=0.002) and cribriform pattern (HR 3.229, 95% CI 1.206 to 8.646, p=0.020) were still independent predictors of OS. STAS (HR 2.091, 95% CI 1.025 to 4.268, Pp=0.034) was a negative indicator of DFS as determined in multivariate analyses (table 4).
Immunohistochemistry and gene mutations
Patients with cribriform patterns in the Aci-predominant subcategory (n=79) expressed the diagnostic markers TTF-1 (55, 69.6%), CK-7 (68, 86.1%) and Napsin-A (53, 67.1%). Seventy-six cases were available for molecular data assessment of anaplastic lymphoma receptor tyrosine kinase (ALK) rearrangements by immunostaining; of these, 8 (10.1%) were positive. Further, three (3.8%) and four (5.1%) cases were positive for epidermal growth factor receptor(EGFR) and kirsten rat sarcoma viral oncogene homolog (KRAS) mutations, respectively, as determined by PCR. Cribriform pattern tumours were not correlated with ALK expression or with EGFR or KRAS mutations.
In 2015, the WHO updated its classification of lung tumours3 as proposed by the IASLC/ATS/ERS.4 The cribriform pattern was classified as an Aci-predominant subtype of lung ADC.3 There is some debate about the prognostic utility of cribriform structures in lung ADC, although several studies have reported correlations between the cribriform pattern and prognosis of ADC in various organs.17–21 Maeshima et al 22 provided the first evidence that the cribriform pattern is associated with a poor outcome in lung ADC. Subsequently, Warth et al 10 noted that the OS of patients with cribriform predominance varied between patients with Aci-predominant and Sol-predominant ADCs. Our findings highlight the important prognostic value of cribriform morphology.
We found slightly higher proportions of cribriform pattern (39.8%) and predominance (11.8%) than those reported by Qu et al 11 (33% with cribriform pattern and 8% with cribriform predominance in a series of 395 stage I–III cases, respectively). These differences may have been due to our definition of cribriform structures, which not ony was limited to back-to-back fusion glands but also included variant glomerular and beaded structures. Consistent with the results of Qu et al 11 and Warth et al ,10 the cribriform pattern was more common in Aci, Sol and Mip subtypes, and was rarely found in the Lep subtype. These results overwhelmingly suggest that cribriform morphology always exists in growth patterns of poor differentiation. Furthermore, the presence of the cribriform pattern was associated with aggressive histological behaviours, such as tumour invasiveness (lymphatic invasion, larger tumour size and metastasis), advanced clinical stages, a higher degree of atypia and proliferative activity. In the Aci-predominant cohort (n=117), patients with cribriform architecture had poorer OS than those without this pattern. Recently, Kadota et al 23 reported that both 5-year OS and DFS were lower in patients with the cribriform subtype than in patients with Aci and Pap subtypes. Kuang et al 24 noted that some complex glandular patterns, including cribriform and fused gland, showed pathological features distinct from the five main subtypes of ADC (Aci, Sol, Mip, Pap and Lep). With regard to the cut-off for cribriform pattern, a previous study showed that cribriform pattern (≥10%) can be used to stratify Aci-predominant tumours with regard to recurrence.9Furthermore, after excluding the cribriform-predominant population (n=33) from the Aci group (n=117), the cribriform pattern was still a risk factor for OS and DFS in the present study.
Patients with the Lep subtype have a higher rate of survival and a lower rate of recurrence, while patients with the Sol and Mip subtypes show poorer survival and higher recurrence rates.7 25–28 In the present study, OS was significantly lower in patients with cribriform-predominant ADC than in those with Aci-predominant or Pap-predominant ADC. However, it was comparable among patients with Mip-predominant or Sol-predominant ADC.7 29 These results suggest that the cribriform pattern might be better grouped in high-grade tumours with Sol-predominant and Mip-predominant ADCs and not into the Aci group, as currently proposed.23
Mitotic count was a risk factor for poor OS in both the Aci-predominant cohort (n=117) and the pure Aci-predominant cohort (n=84). Previously, the effects of mitotic activity on prognosis were verified in invasive ADC of the lung.30 However, atypia was not a risk factor for OS or DFS, probably because the nuclei of carcinoma cells are generally atypical in the invasive lung ADC. Furthermore, the presence of STAS in Aci-predominant ADC has negative prognostic significance in terms of DFS, which means that STAS is a predictive factor for relapses in lung ADC.31 32
Similar to previous studies, no correlations were identified between cribriform pattern and EGFR mutations, KRAS mutations or ALK rearrangements in the Aci-predominant group.9 One limitation of this finding is that ALK rearrangement was not confirmed by PCR or fluorescence in situ hybridisation. ALK rearrangements were significantly characterised by the Sol-predominant subtype with mucin production, the cribriform pattern and a signet-ring cell appearance.29 Although previous reports have demonstrated that ALK and ROS1 gene rearrangements occur frequently in young, non-smoking patients with cribriform, Sol or signet-ring cell predominance, these features have no specific role in selecting patients for molecular testing. Instead, molecular testing is recommended in patients with lung cancer with ADC and in patients with mixed lung cancer with ADC.33 These differential results may be attributable to the limited availability of molecular data, non-uniform detection conditions or due to differences between populations.
There were some limitations to this study. Similar to other retrospective, observational cohort studies at single institutions, there was the potential for referral and selection bias. In addition, given the relatively small number of patients, larger studies with sufficient statistical power are needed to validate the associations between cribriform cancer and clinicopathological characteristics, gene mutations and clinical outcomes of patients with lung cancer.
The cribriform pattern is associated with aggressive pathological behaviours, including advanced stages of cancer, lymph node invasion, metastasis and larger tumour sizes. In addition, it is an independent factor of poor prognosis in patients with resected Aci-predominant ADC.
Take home messages
The paper reveals that cribriform pattern not only includes back-to-back fused tumour glands, but glomeruloid pattern beaded pattern. Cribriform pattern is an independent factor for poor prognosis in patients with resected Aci-predominant adenocarcinoma (ADC). In addition, the outcome of the cribriform-predominant subtype was poorer than the acinar-predominant subtype. Therefore, when a pathologist diagnoses ADC of the lung, the percentage of cribriform should be noted. Clinicians should understand that patients with a cribriform pattern have a poor prognosis. The paper also reveals that the cribriform pattern should be considered a new histologic subtype of adenocarcinoma of the lung and that it should be rather grouped in the high-grade tumors together with solid and micropapillary predominant adenocarcinomas.
Handling editor Runjan Chetty.
Contributors The corresponding authors directed the research. RZ completed the main article framework and written documents. GH retrospectively analysed the pathological sections. JQ provided clinical information. HW analysed the date. The other authors revised the paper.
Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.
Competing interests None declared.
Patient consent for publication Parental/guardian consent obtained.
Provenance and peer review Not commissioned; externally peer reviewed.
Data availability statement Data are available in a public, open access repository. Data are available upon reasonable request. Data may be obtained from a third party and are not publicly available. All data relevant to the study are included in the article or uploaded as supplementary information.